Insecticidal proteins and methods for their use

ABSTRACT

Compositions and methods for controlling pests are provided. The methods involve transforming organisms with a nucleic acid sequence encoding an insecticidal protein. In particular, the nucleic acid sequences are useful for preparing plants and microorganisms that possess insecticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. Compositions are insecticidal nucleic acids and proteins of bacterial species. The sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest including plants, as probes for the isolation of other homologous (or partially homologous) genes. The pesticidal proteins find use in controlling, inhibiting growth or killing Lepidopteran, Coleopteran, Dipteran, fungal, Hemipteran and nematode pest populations and for producing compositions with insecticidal activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit of U.S. Provisional Application No.62/331,708, filed on May 4, 2016, which is incorporated herein byreference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“6441WOPCT_Sequence_Listing” created on May 1, 2017, and having a sizeof 1,094 kilobytes and is filed concurrently with the specification. Thesequence listing contained in this ASCII formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

FIELD

This disclosure relates to the field of molecular biology. Provided arenovel genes that encode pesticidal proteins. These pesticidal proteinsand the nucleic acid sequences that encode them are useful in preparingpesticidal formulations and in the production of transgenicpest-resistant plants.

BACKGROUND

Biological control of insect pests of agricultural significance using amicrobial agent, such as fungi, bacteria or another species of insectaffords an environmentally friendly and commercially attractivealternative to synthetic chemical pesticides. Generally speaking, theuse of biopesticides presents a lower risk of pollution andenvironmental hazards and biopesticides provide greater targetspecificity than is characteristic of traditional broad-spectrumchemical insecticides. In addition, biopesticides often cost less toproduce and thus improve economic yield for a wide variety of crops.

Certain species of microorganisms of the genus Bacillus are known topossess pesticidal activity against a range of insect pests includingLepidoptera, Diptera, Coleoptera, Hemiptera and others. Bacillusthuringiensis (Bt) and Bacillus popilliae are among the most successfulbiocontrol agents discovered to date. Insect pathogenicity has also beenattributed to strains of B. larvae, B. lentimorbus, B. sphaericus and B.cereus. Microbial insecticides, particularly those obtained fromBacillus strains, have played an important role in agriculture asalternatives to chemical pest control.

Crop plants have been developed with enhanced insect resistance bygenetically engineering crop plants to produce pesticidal proteins fromBacillus. For example, corn and cotton plants have been geneticallyengineered to produce pesticidal proteins isolated from strains ofBacillus thuringiensis. These genetically engineered crops are nowwidely used in agriculture and have provided the farmer with anenvironmentally friendly alternative to traditional insect-controlmethods. While they have proven to be very successful commercially,these genetically engineered, insect-resistant crop plants provideresistance to only a narrow range of the economically important insectpests. In some cases, insects can develop resistance to differentinsecticidal compounds, which raises the need to identify alternativebiological control agents for pest control.

Accordingly, there remains a need for new pesticidal proteins withdifferent ranges of insecticidal activity against insect pests, e.g.,insecticidal proteins which are active against a variety of insects inthe order Lepidoptera and the order Coleoptera including but not limitedto insect pests that have developed resistance to existing insecticides.

SUMMARY

In one aspect compositions and methods for conferring pesticidalactivity to bacteria, plants, plant cells, tissues and seeds areprovided. Compositions include nucleic acid molecules encoding sequencesfor pesticidal and insecticidal polypeptides, vectors comprising thosenucleic acid molecules, and host cells comprising the vectors.Compositions also include the pesticidal polypeptide sequences andantibodies to those polypeptides. Compositions also comprise transformedbacteria, plants, plant cells, tissues and seeds.

In another aspect isolated or recombinant nucleic acid molecules areprovided encoding IPD090 polypeptides including amino acidsubstitutions, deletions, insertions, and fragments thereof. Providedare isolated or recombinant nucleic acid molecules capable of encodingIPD090 polypeptides of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 379 or SEQ ID NO: 384, as well as amino acid substitutions,deletions, insertions, fragments thereof, and combinations thereof.Nucleic acid sequences that are complementary to a nucleic acid sequenceof the embodiments or that hybridize to a sequence of the embodimentsare also encompassed. The nucleic acid sequences can be used in DNAconstructs or expression cassettes for transformation and expression inorganisms, including microorganisms and plants. The nucleotide or aminoacid sequences may be synthetic sequences that have been designed forexpression in an organism including, but not limited to, a microorganismor a plant.

In another aspect IPD090 polypeptides are encompassed. Also provided areisolated or recombinant IPD090 polypeptides of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 379, and SEQ ID NO: 384, as well as aminoacid substitutions, deletions, insertions, fragments thereof andcombinations thereof.

In another aspect, methods are provided for producing the polypeptidesand for using those polypeptides for controlling or killing aLepidopteran, Coleopteran, nematode, fungi, and/or Dipteran pests. Thetransgenic plants of the embodiments express one or more of thepesticidal sequences disclosed herein. In various embodiments, thetransgenic plant further comprises one or more additional genes forinsect resistance, for example, one or more additional genes forcontrolling Coleopteran, Lepidopteran, Hemipteran or nematode pests. Itwill be understood by one of skill in the art that the transgenic plantmay comprise any gene imparting an agronomic trait of interest.

In another aspect, methods for detecting the nucleic acids andpolypeptides of the embodiments in a sample are also included. A kit fordetecting the presence of an IPD090 polypeptide or detecting thepresence of a polynucleotide encoding an IPD090 polypeptide in a sampleis provided. The kit may be provided along with all reagents and controlsamples necessary for carrying out a method for detecting the intendedagent, as well as instructions for use.

In another aspect the compositions and methods of the embodiments areuseful for the production of organisms with enhanced pest resistance ortolerance. These organisms and compositions comprising the organisms aredesirable for agricultural purposes. The compositions of the embodimentsare also useful for generating altered or improved proteins that havepesticidal activity or for detecting the presence of IPD090polypeptides.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1B shows an amino acid sequence alignment, using the ALIGNX®module of the Vector NTI® suite, of the IPD090Aa polypeptide (SEQ ID NO:2) and the IPD090Ca polypeptide (SEQ ID NO: 6). The amino acid sequencediversity between the amino acid sequences is highlighted. Conservativeamino acid differences are indicated by (

) shading and non-conservative amino acid difference by (

) shading. The N-terminal amino acids deleted compared to the IPD090Aapolypeptide (SEQ ID NO: 2) in the truncation variant, IPD090Aa (TR1)polypeptide (SEQ ID NO: 10) of Example 6, are underlined in the IPD090Aasequence (SEQ ID NO: 2). The respective boundary points of theIPD090Aa/IPD090Ca chimera proteins of Example 11 are indicated below theIPD090Aa sequence (SEQ ID NO: 2) by a “▴” above the IPD090Ca sequence(SEQ ID NO: 6) by a “▾”.

FIG. 2 shows a bar chart reflecting densitometry values of in-gelfluorescence, from the SDS-PAGE gel of Example 14, for homologouscompetition of 6.3 nM IPD090Aa^(Alexa) binding to WCRW BBMVs normalizedto the binding signal in the absence of unlabeled protein (“6 nMAlexa-IPD090”) and in the presence of a saturating concentration ofunlabeled protein (“+13 μM IPD090”). The difference in magnitude betweenthe bars reflects specific binding.

FIG. 3 shows the corn rootworm node injury score (CRWNIS) Score ofindividual transgenic T0 maize events from constructs PHP73234 andPHP73237 expressing the IPD090 polypeptide of SEQ ID NO: 377, andPHP77372 expressing the IPD090 polypeptide of SEQ ID NO: 10.

FIG. 4 shows the structure of IPD090Aa (SEQ ID NO: 2), as determined byX-ray Crystallography, indicating the MACPF domain of the N-terminalregion and a 3-prism domain of the C-terminal domain region. A Mg+ atomis shown as a sphere at the bottom of the β-prism domain. The twoclusters of helices are labeled as CH1 and CH2.

FIG. 5 shows a 90-degree rotation of the IPD090Aa polypeptide (SEQ IDNO: 2) about the vertical axis showing the N-terminal β-strand for a 5thmember of the central β-sheet.

FIG. 6 shows a close up view of the C-terminal β-prism domain of theIPD090Aa (SEQ ID NO: 2) structure illustrating the 3-fold axis and Mg+2interactions.

FIG. 7 shows the Ramachadran Plot for refined IPD090Aa (SEQ ID NO: 2)2.1 Å structure.

DETAILED DESCRIPTION

It is to be understood that this disclosure is not limited to theparticular methodology, protocols, cell lines, genera, and reagentsdescribed, as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentdisclosure.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisdisclosure belongs unless clearly indicated otherwise.

The present disclosure is drawn to compositions and methods forcontrolling pests. The methods involve transforming organisms withnucleic acid sequences encoding IPD090 polypeptides. In particular, thenucleic acid sequences of the embodiments are useful for preparingplants and microorganisms that possess pesticidal activity. Thus,transformed bacteria, plants, plant cells, plant tissues and seeds areprovided. The compositions include pesticidal nucleic acids and proteinsof bacterial species. The nucleic acid sequences find use in theconstruction of expression vectors for subsequent transformation intoorganisms of interest, as probes for the isolation of other homologous(or partially homologous) genes, and for the generation of alteredIPD090 polypeptides by methods known in the art, such as site directedmutagenesis, domain swapping or DNA shuffling. The IPD090 polypeptidesfind use in controlling or killing Lepidopteran, Coleopteran, Dipteran,fungal, Hemipteran and nematode pest populations and for producingcompositions with pesticidal activity. Insect pests of interest include,but are not limited to, Lepidoptera species including but not limitedto: Corn Earworm, (CEW) (Helicoverpa zea), European Corn Borer (ECB)(Ostrinia nubialis), diamond-back moth, e.g., Helicoverpa zea Boddie;soybean looper, e.g., Pseudoplusia includens Walker; and velvet beancaterpillar e.g., Anticarsia gemmatalis Hübner and Coleoptera speciesincluding but not limited to Western corn rootworm (Diabroticavirgifera)—WCRW, Southern corn rootworm (Diabrotica undecimpunctatahowardi)—SCRW, and Northern corn rootworm (Diabrotica barberi)—NCRW.

By “pesticidal toxin” or “pesticidal protein” is used herein to refer toa toxin that has toxic activity against one or more pests, including,but not limited to, members of the Lepidoptera, Diptera, Hemiptera andColeoptera orders or the Nematoda phylum or a protein that has homologyto such a protein. Pesticidal proteins have been isolated from organismsincluding, for example, Bacillus sp., Pseudomonas sp., Photorhabdus sp.,Xenorhabdus sp., Clostridium bifermentans and Paenibacillus popilliae.Pesticidal proteins include but are not limited to: insecticidalproteins from Pseudomonas sp. such as PSEEN3174 (Monalysin; (2011) PLoSPathogens 7:1-13); from Pseudomonas protegens strain CHAO and Pf-5(previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology10:2368-2386; GenBank Accession No. EU400157); from Pseudomonastaiwanensis (Liu, et al., (2010) J. Agric. Food Chem., 58:12343-12349)and from Pseudomonas pseudoalcaligenes (Zhang, et al., (2009) Annals ofMicrobiology 59:45-50 and Li, et al., (2007) Plant Cell Tiss. OrganCult. 89:159-168); insecticidal proteins from Photorhabdus sp. andXenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxicology Journal,3:101-118 and Morgan, et al., (2001) Applied and Envir. Micro.67:2062-2069); U.S. Pat. Nos. 6,048,838, and 6,379,946; a PIP-1polypeptide of US Patent Publication US20140007292; an AflP-1A and/orAflP-1B polypeptide of U.S. Pat. No. 9,475,847; a PHI-4 polypeptide ofUS Patent Publication US20140274885 and US20160040184; a PIP-47polypeptide of US Publication Number US20160186204, a PIP-72 polypeptideof US Patent Publication Number US20160366891; a PtlP-50 polypeptide anda PtlP-65 polypeptide of PCT Publication Number WO2015/120270; a PtlP-83polypeptide of PCT Publication Number WO2015/120276; a PtlP-96polypeptide of PCT Serial Number PCT/US15/55502; an IPD079 polypeptideof PCT Publication Number WO2017/23486; an IPD082 polypeptide of SerialNumber PCT/US16/65531, an IPD093 polypeptide of U.S. Ser. No.62/434,020; an IPD080 polypeptide of U.S. Ser. No. U.S. 62/411,318; andδ-endotoxins including, but not limited to, the Cry1, Cry2, Cry3, Cry4,Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry15,Cry16, Cry17, Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24, Cry25,Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, Cry33, Cry34, Cry35,Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45,Cry46, Cry47, Cry49, Cry50, Cry51, Cry52, Cry53, Cry54, Cry55, Cry56,Cry57, Cry58, Cry59, Cry60, Cry61, Cry62, Cry63, Cry64, Cry65, Cry66,Cry67, Cry68, Cry69, Cry70, Cry71, and Cry72 classes of δ-endotoxingenes and the B. thuringiensis cytolytic cyt1 and cyt2 genes. Members ofthese classes of B. thuringiensis insecticidal proteins well known toone skilled in the art (see, Crickmore, et al., “Bacillus thuringiensistoxin nomenclature” (2011), atlifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can be accessed onthe world-wide web using the “www” prefix).

Examples of δ-endotoxins also include but are not limited to Cry1Aproteins of U.S. Pat. Nos. 5,880,275 and 7,858,849; a Cry1Ac mutant ofU.S. Pat. No. 9,512,187; a DIG-3 or DIG-11 toxin (N-terminal deletion ofα-helix 1 and/or α-helix 2 variants of cry proteins such as Cry1A,Cry3A) of U.S. Pat. Nos. 8,304,604, 8,304,605 and 8,476,226; Cry1B ofU.S. patent application Ser. No. 10/525,318, US Patent ApplicationPublication Number US20160194364, and U.S. Pat. Nos. 9,404,121 and8,772,577; Cry1B variants of PCT Publication Number WO2016/61197 andSerial Number PCT/US17/27160; Cry1C of U.S. Pat. No. 6,033,874; Cry1F ofU.S. Pat. Nos. 5,188,960 and 6,218,188; Cry1A/F chimeras of U.S. Pat.Nos. 7,070,982; 6,962,705 and 6,713,063); a Cry2 protein such as Cry2Abprotein of U.S. Pat. No. 7,064,249); a Cry3A protein including but notlimited to an engineered hybrid insecticidal protein (eHIP) created byfusing unique combinations of variable regions and conserved blocks ofat least two different Cry proteins (US Patent Application PublicationNumber 2010/0017914); a Cry4 protein; a Cry5 protein; a Cry6 protein;Cry8 proteins of U.S. Pat. Nos. 7,329,736, 7,449,552, 7,803,943,7,476,781, 7,105,332, 7,339,092, 7,378,499, 7,462,760, and 9,593,345; aCry9 protein such as such as members of the Cry9A, Cry9B, Cry9C, Cry9D,Cry9E and Cry9F families including the Cry9 protein of U.S. Pat. Nos.9,000,261 and 8,802,933, and U.S. Ser. No. 62/287,281; a Cry15 proteinof Naimov, et al., (2008) Applied and Environmental Microbiology,74:7145-7151; a Cry14 protein of U.S. Pat. No. 8,933,299; a Cry22, aCry34Ab1 protein of U.S. Pat. Nos. 6,127,180, 6,624,145 and 6,340,593; atruncated Cry34 protein of U.S. Pat. No. 8,816,157; a CryET33 andcryET34 protein of U.S. Pat. Nos. 6,248,535, 6,326,351, 6,399,330,6,949,626, 7,385,107 and 7,504,229; a CryET33 and CryET34 homologs of USPatent Publication Number 2006/0191034, 2012/0278954, and PCTPublication Number WO 2012/139004; a Cry35Ab1 protein of U.S. Pat. Nos.6,083,499, 6,548,291 and 6,340,593; a Cry46 protein of U.S. Pat. No.9,403,881, a Cry51 protein, a Cry binary toxin; a TIC901 or relatedtoxin; TIC807 of US Patent Application Publication Number 2008/0295207;TIC853 of US Patent U.S. Pat. No. 8,513,493; ET29, ET37, TIC809, TIC810,TIC812, TIC127, TIC128 of PCT US 2006/033867; engineered Hemipterantoxic proteins of US Patent Application Publication NumberUS20160150795, AXMI-027, AXMI-036, and AXMI-038 of U.S. Pat. No.8,236,757; AXMI-031, AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. No.7,923,602; AXMI-018, AXMI-020 and AXMI-021 of WO 2006/083891; AXMI-010of WO 2005/038032; AXMI-003 of WO 2005/021585; AXMI-008 of US PatentApplication Publication Number 2004/0250311; AXMI-006 of US PatentApplication Publication Number 2004/0216186; AXMI-007 of US PatentApplication Publication Number 2004/0210965; AXMI-009 of US PatentApplication Number 2004/0210964; AXMI-014 of US Patent ApplicationPublication Number 2004/0197917; AXMI-004 of US Patent ApplicationPublication Number 2004/0197916; AXMI-028 and AXMI-029 of WO2006/119457; AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 andAXMI-004 of WO 2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416;AXMI-205 of US Patent Application Publication Number 2011/0023184;AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037,AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063 andAXMI-064 of US Patent Application Publication Number 2011/0263488;AXMI046, AXMI048, AXMI050, AXMI051, AXMI052, AXMI053, AXMI054, AXMI055,AXMI056, AXMI057, AXMI058, AXMI059, AXMI060, AXMI061, AXMI067, AXMI069,AXMI071, AXMI072, AXMI073, AXMI074, AXMI075, AXMI087, AXMI088, AXMI093,AXMI070, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097,AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107,AXMI108, AXMI109, AXMI110, AXMI111, AXMI112, AXMI114, AXMI116, AXMI117,AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI125,AXMI126, AXMI127, AXMI129, AXMI151, AXMI161, AXMI164, AXMI183, AXMI132,AXMI137, AXMI138 of US Patent U.S. Pat. Nos. 8,461,421 and 8,461,422;AXMI-R1 and related proteins of US Patent Application Publication Number2010/0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z and AXMI225z of WO2011/103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228,AXMI229, AXMI230 and AXMI231 of WO 2011/103247; AXMI-115, AXMI-113,AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. 8,334,431; AXMI-001,AXMI-002, AXMI-030, AXMI-035 and AXMI-045 of US Patent ApplicationPublication Number 2010/0298211; AXMI-066 and AXMI-076 of US PatentApplication Publication Number 2009/0144852; AXMI128, AXMI130, AXMI131,AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148,AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158,AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171,AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179,AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189of U.S. Pat. No. 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091,AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102,AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, dsAXMI111,AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121,AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129,AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of USPatent U.S. Pat. No. 8,461,421; AXMI192 of US Patent U.S. Pat. No.8,461,415; AXMI281 of US Patent Application Publication NumberUS20160177332; AXMI422 of U.S. Pat. No. 8,252,872; cry proteins such asCry1A and Cry3A having modified proteolytic sites of U.S. Pat. No.8,319,019; a Cry1Ac, Cry2Aa and Cry1Ca toxin protein from Bacillusthuringiensis strain VBTS 2528 of US Patent Application PublicationNumber 2011/0064710. The Cry proteins MP032, MP049, MP051, MP066, MP068,MP070, MP091S, MP109S, MP114, MP121, MP134S, MP183S, MP185S, MP186S,MP195S, MP197S, MP208S, MP209S, MP212S, MP214S, MP217S, MP222S, MP234S,MP235S, MP237S, MP242S, MP243, MP248, MP249S, MP251M, MP252S, MP253,MP259S, MP287S, MP288S, MP295S, MP296S, MP297S, MP300S, MP304S, MP306S,MP310S, MP312S, MP314S, MP319S, MP325S, MP326S, MP327S, MP328S, MP334S,MP337S, MP342S, MP349S, MP356S, MP359S, MP360S, MP437S, MP451S, MP452S,MP466S, MP468S, MP476S, MP482S, MP522S, MP529S, MP548S, MP552S, MP562S,MP564S, MP566S, MP567S, MP569S, MP573S, MP574S, MP575S, MP581S, MP590,MP594S, MP596S, MP597, MP599S, MP600S, MP601S, MP602S, MP604S, MP626S,MP629S, MP630S, MP631S, MP632S, MP633S, MP634S, MP635S, MP639S, MP640S,MP644S, MP649S, MP651S, MP652S, MP653S, MP661S, MP666S, MP672S, MP696S,MP704S, MP724S, MP729S, MP739S, MP755S, MP773S, MP799S, MP800S, MP801S,MP802S, MP803S, MP805S, MP809S, MP815S, MP828S, MP831S, MP844S, MP852,MP865S, MP879S, MP887S, MP891S, MP896S, MP898S, MP935S, MP968, MP989,MP993, MP997, MP1049, MP1066, MP1067, MP1080, MP1081, MP1200, MP1206,MP1233, and MP1311 of U.S. Ser. No. 62/429,426. The insecticidalactivity of Cry proteins is well known to one skilled in the art (forreview, see, van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16). Theuse of Cry proteins as transgenic plant traits is well known to oneskilled in the art and Cry-transgenic plants including but not limitedto plants expressing Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F,Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1,Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatoryapproval (see, Sanahuja, (2011) Plant Biotech Journal 9:283-300 and theCERA. (2010) GM Crop Database Center for Environmental Risk Assessment(CERA), ILSI Research Foundation, Washington D.C. atcera-gmc.org/index.php?action=gm_crop_database which can be accessed onthe world-wide web using the “www” prefix). More than one pesticidalproteins well known to one skilled in the art can also be expressed inplants such as Vip3Ab & Cry1Fa (US2012/0317682); Cry1BE & Cry1F(US2012/0311746); Cry1CA & Cry1AB (US2012/0311745); Cry1F & CryCa(US2012/0317681); Cry1 DA & Cry1BE (US2012/0331590); Cry1 DA & Cry1Fa(US2012/0331589); Cry1AB & Cry1BE (US2012/0324606); Cry1Fa & Cry2Aa andCry1I & Cry1E (US2012/0324605); Cry34Ab/35Ab and Cry6Aa (US20130167269);Cry34Ab/VCry35Ab & Cry3Aa (US20130167268); and Cry3A and Cry1Ab orVip3Aa (U.S. Pat. No. 9,045,766). Pesticidal proteins also includeinsecticidal lipases including lipid acyl hydrolases of U.S. Pat. No.7,491,869, and cholesterol oxidases such as from Streptomyces (Purcellet al. (1993) Biochem Biophys Res Commun 15:1406-1413). Pesticidalproteins also include VIP (vegetative insecticidal proteins) toxins ofU.S. Pat. Nos. 5,877,012, 6,107,279 6,137,033, 7,244,820, 7,615,686, and8,237,020 and the like. Other VIP proteins are well known to one skilledin the art (see, lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.htmlwhich can be accessed on the world-wide web using the “www” prefix).Pesticidal proteins also include toxin complex (TC) proteins, obtainablefrom organisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see,U.S. Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have “standalone” insecticidal activity and other TC proteins enhance the activityof the stand-alone toxins produced by the same given organism. Thetoxicity of a “stand-alone” TC protein (from Photorhabdus, Xenorhabdusor Paenibacillus, for example) can be enhanced by one or more TC protein“potentiators” derived from a source organism of a different genus.There are three main types of TC proteins. As referred to herein, ClassA proteins (“Protein A”) are stand-alone toxins. Class B proteins(“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity ofClass A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 andXptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi.Examples of Class C proteins are TccC, XptC1Xb and XptB1Wi. Pesticidalproteins also include spider, snake and scorpion venom proteins.Examples of spider venom peptides include but not limited to lycotoxin-1peptides and mutants thereof (U.S. Pat. No. 8,334,366).

In some embodiments the IPD090 polypeptide includes an amino acidsequence deduced from the full-length nucleic acid sequence disclosedherein and amino acid sequences that are shorter than the full-lengthsequences, either due to the use of an alternate downstream start siteor due to processing that produces a shorter protein having pesticidalactivity. Processing may occur in the organism the protein is expressedin or in the pest after ingestion of the protein.

Thus, provided herein are novel isolated or recombinant nucleic acidsequences that confer pesticidal activity. Also provided are the aminoacid sequences of IPD090 polypeptides. The protein resulting fromtranslation of these IPD090 genes allows cells to control or kill peststhat ingest it.

IPD090 Proteins and Variants and Fragments Thereof

IPD090 polypeptides are encompassed by the disclosure. “IPD090polypeptide”, and “IPD090 protein” as used herein interchangeably refersto a polypeptide having insecticidal activity including but not limitedto insecticidal activity against one or more insect pests of theLepidoptera and/or Coleoptera orders, and is sufficiently homologous tothe IPD090Aa polypeptide of SEQ ID NO: 2. A variety of IPD090polypeptides are contemplated. Sources of IPD090 polypeptides or relatedproteins include bacterial species selected from but not limited toPseudomonas species and Woodsholea species. Alignment of the amino acidsequences of IPD090 polypeptide homologs (for example—FIG. 1), allowsfor the identification of residues that are highly conserved amongst thenatural homologs of this family.

“Sufficiently homologous” is used herein to refer to an amino acidsequence that has at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence homology compared to a reference sequenceusing one of the alignment programs described herein using standardparameters. In some embodiments the sequence homology is against thefull length sequence of an IPD090 polypeptide. In some embodiments theIPD090 polypeptide has at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or greater sequence identity compared to SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379 or SEQ ID NO: 384. The term“about” when used herein in context with percent sequence identitymeans+/−0.5%. One of skill in the art will recognize that these valuescan be appropriately adjusted to determine corresponding homology ofproteins taking into account amino acid similarity and the like. In someembodiments the sequence identity is calculated using ClustalW algorithmin the ALIGNX® module of the Vector NTI® Program Suite (InvitrogenCorporation, Carlsbad, Calif.) with all default parameters. In someembodiments the sequence identity is across the entire length ofpolypeptide calculated using ClustalW algorithm in the ALIGNX® module ofthe Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, Calif.)with all default parameters.

As used herein, the terms “protein,” “peptide molecule,” or“polypeptide” includes any molecule that comprises five or more aminoacids. It is well known in the art that protein, peptide or polypeptidemolecules may undergo modification, including post-translationalmodifications, such as, but not limited to, disulfide bond formation,glycosylation, phosphorylation or oligomerization. Thus, as used herein,the terms “protein,” “peptide molecule” or “polypeptide” includes anyprotein that is modified by any biological or non-biological process.The terms “amino acid” and “amino acids” refer to all naturallyoccurring L-amino acids.

A “recombinant protein” is used herein to refer to a protein that is nolonger in its natural environment, for example in vitro or in arecombinant bacterial or plant host cell. An IPD090 polypeptide that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10% or 5% (by dry weight) ofnon-pesticidal protein (also referred to herein as a “contaminatingprotein”).

“Fragments” or “biologically active portions” include polypeptidefragments comprising amino acid sequences sufficiently identical to anIPD090 polypeptide and that exhibit insecticidal activity. “Fragments”or “biologically active portions” of IPD090 polypeptides includesfragments comprising amino acid sequences sufficiently identical to theamino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO: 379 or SEQ ID NO: 384 wherein the IPD090 polypeptide hasinsecticidal activity. Such biologically active portions can be preparedby recombinant techniques and evaluated for insecticidal activity. Insome embodiments, the IPD090 polypeptide fragment is an N-terminaland/or a C-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31 or more amino acids from the N-terminus and/or C-terminusrelative to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, SEQ ID NO: 379or SEQ ID NO: 384, e.g., by proteolysis, by insertion of a start codon,by deletion of the codons encoding the deleted amino acids andconcomitant insertion of a start codon, and/or insertion of a stopcodon. In some embodiments, the IPD090 polypeptide fragment is anN-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 amino acids from theN-terminus of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379or SEQ ID NO: 384. In some embodiments, the IPD090 polypeptide fragmentis an N-terminal and/or a C-terminal truncation of at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or more amino acids from theN-terminus and/or C-terminus relative to SEQ ID NO: 2, SEQ ID NO: 4 orSEQ ID NO: 6, SEQ ID NO: 379 or SEQ ID NO: 384. In some embodiments, theIPD090 polypeptide fragment comprises amino acids 1-315, amino acids1-330, amino acids 1-349, amino acids 1-450, amino acids 25-315, aminoacids 25-330, amino acids 25-349, amino acids 25-450 or amino acids25-483 of any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:379 or SEQ ID NO: 384. In some embodiments the truncated variant is thepolypeptide of SEQ ID NO: 10.

“Variants” as used herein refers to proteins or polypeptides having anamino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater identical to the parental aminoacid sequence.

In some embodiments an IPD090 polypeptide comprises an amino acidsequence having at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater identity to the amino acid sequence of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379 or SEQ ID NO: 384, whereinthe IPD090 polypeptide has insecticidal activity.

In some embodiments an IPD090 polypeptide comprises an amino acidsequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greateridentity across the entire length of the amino acid sequence of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379 or SEQ ID NO: 384.

In some embodiments the sequence identity is across the entire length ofthe polypeptide calculated using ClustalW algorithm in the ALIGNX®module of the Vector NTI® Program Suite (Invitrogen Corporation,Carlsbad, Calif.) with all default parameters.

In some embodiments the IPD090 polypeptide comprises an amino acidsequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greateridentity across the entire length of the amino acid sequence of SEQ IDNO: 2.

In some embodiments an IPD090 polypeptide comprises an amino acidsequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379 orSEQ ID NO: 384 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95 or more amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379 or SEQ ID NO: 384.

In some embodiments an IPD090 polypeptide variant comprises any one ormore amino acid substitutions corresponding to positions 3, 4, 8, 12,15, 16, 21, 23, 24, 26, 28, 30, 38, 46, 47, 50, 52, 55, 62, 63, 67, 68,70, 73, 74, 75, 76, 80, 90, 91, 94, 99, 100, 108, 115, 127, 129, 161,169, 175, 177, 178, 180, 185, 207, 213, 223, 240, 241, 247, 255, 266,273, 275, 277, 278, 287, 288, 302, 306, 309, 310, 311, 312, 316, 317,318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331,332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345,346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359,360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373,374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387,388, 389, 391, 392, 395, 397, 400, 401, 402, 405, 407, 410, 423, 425,426, 431, 433, 434, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446,447, 448, 449, 450, 451, 452, 453, 454, 455, 457, 458, 459, 460, 468,and 471 of SEQ ID NO: 2, in any combination.

In some embodiments an IPD090 polypeptide variant comprises any one ormore active amino acid substitutions of Table 10 and/or 12.

Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of an IPD090 polypeptide can beprepared by mutations in the DNA. This may also be accomplished by oneof several forms of mutagenesis and/or in directed evolution. In someaspects, the changes encoded in the amino acid sequence will notsubstantially affect the function of the protein. Such variants willpossess the desired pesticidal activity. However, it is understood thatthe ability of an IPD090 polypeptide to confer pesticidal activity maybe improved by the use of such techniques upon the compositions of thisdisclosure.

For example, conservative amino acid substitutions may be made at one ormore predicted nonessential amino acid residues. A “nonessential” aminoacid residue is a residue that can be altered from the wild-typesequence of an IPD090 polypeptide without altering the biologicalactivity. Nonessential amino acid residues can be identified by aligningrelated IPD090 homologs such as is shown in FIG. 1. A “conservativeamino acid substitution” is one in which the amino acid residue isreplaced with an amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have beendefined in the art. These families include: amino acids with basic sidechains (e.g., lysine, arginine, histidine); acidic side chains (e.g.,aspartic acid, glutamic acid); polar, negatively charged residues andtheir amides (e.g., aspartic acid, asparagine, glutamic, acid,glutamine; uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine); small aliphatic,nonpolar or slightly polar residues (e.g., Alanine, serine, threonine,proline, glycine); nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); largealiphatic, nonpolar residues (e.g., methionine, leucine, isoleucine,valine, cystine); beta-branched side chains (e.g., threonine, valine,isoleucine); aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine); large aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan).

Amino acid substitutions may be made in nonconserved regions that retainfunction. In general, such substitutions would not be made for conservedamino acid residues or for amino acid residues residing within aconserved motif, where such residues are essential for protein activity.Examples of residues that are conserved and that may be essential forprotein activity include, for example, residues that are identicalbetween all proteins contained in an alignment of similar or relatedtoxins to the sequences of the embodiments (e.g., residues that areidentical in an alignment of homologous proteins). Examples of residuesthat are conserved but that may allow conservative amino acidsubstitutions and still retain activity include, for example, residuesthat have only conservative substitutions between all proteins containedin an alignment of similar or related toxins to the sequences of theembodiments (e.g., residues that have only conservative substitutionsbetween all proteins contained in the alignment homologous proteins).However, one of skill in the art would understand that functionalvariants may have minor conserved or nonconserved alterations in theconserved residues. Guidance as to appropriate amino acid substitutionsthat do not affect biological activity of the protein of interest may befound in the model of Dayhoff, et al., (1978) Atlas of Protein Sequenceand Structure (Natl. Biomed. Res. Found., Washington, D.C.), hereinincorporated by reference.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, (1982) J Mol Biol.157(1):105-32). It is accepted that the relative hydropathic characterof the amino acid contributes to the secondary structure of theresultant protein, which in turn defines the interaction of the proteinwith other molecules, for example, enzymes, substrates, receptors, DNA,antibodies, antigens, and the like.

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. Each amino acid has beenassigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte and Doolittle, ibid). These are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9) and arginine(−4.5). In making such changes, the substitution of amino acids whosehydropathic indices are within +2 is preferred, those which are within+1 are particularly preferred, and those within +0.5 are even moreparticularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, states that the greatest local average hydrophilicity ofa protein, as governed by the hydrophilicity of its adjacent aminoacids, correlates with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0.+0.1); glutamate (+3.0.+0.1); serine(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine(−0.4); proline (−0.5.+0.1); alanine (−0.5); histidine (−0.5); cysteine(−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine(−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by virtue of inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein-coding sequences,such as those used commonly in the art to generate protein fusions. Suchfusion proteins are often used to (1) increase expression of a proteinof interest (2) introduce a binding domain, enzymatic activity orepitope to facilitate either protein purification, protein detection orother experimental uses known in the art (3) target secretion ortranslation of a protein to a subcellular organelle, such as theperiplasmic space of Gram-negative bacteria, mitochondria orchloroplasts of plants or the endoplasmic reticulum of eukaryotic cells,the latter of which often results in glycosylation of the protein.

Variant nucleotide and amino acid sequences of the disclosure alsoencompass sequences derived from mutagenic and recombinogenic proceduressuch as DNA shuffling. With such a procedure, one or more differentIPD090 polypeptide coding regions can be used to create a new IPD090polypeptide possessing the desired properties. In this manner, librariesof recombinant polynucleotides are generated from a population ofrelated sequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between a pesticidal geneand other known pesticidal genes to obtain a new gene coding for aprotein with an improved property of interest, such as an increasedinsecticidal activity. Strategies for such DNA shuffling are known inthe art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer, (1994) Nature 370:389-391; Crameri, et al.,(1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol.272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri, et al., (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for generating alteredIPD090 polypeptides. Domains may be swapped between IPD090 polypeptidesresulting in hybrid or chimeric toxins with improved insecticidalactivity or target spectrum. Methods for generating recombinant proteinsand testing them for pesticidal activity are well known in the art (see,for example, Naimov, et al., (2001) Appl. Environ. Microbiol.67:5328-5330; de Maagd, et al., (1996) Appl. Environ. Microbiol.62:1537-1543; Ge, et al., (1991) J. Biol. Chem. 266:17954-17958;Schnepf, et al., (1990) J. Biol. Chem. 265:20923-20930; Rang, et al.,91999) Appl. Environ. Microbiol. 65:2918-2925).

Phylogenetic, sequence motif, and structural analyses of insecticidalprotein families. A sequence and structure analysis method can beemployed, which is composed of four components: phylogenetic treeconstruction, protein sequence motifs finding, secondary structureprediction, and alignment of protein sequences and secondary structures.Details about each component are illustrated below.

1) Phylogenetic Tree Construction

The phylogenetic analysis can be performed using the software MEGA5.Protein sequences can be subjected to ClustalW version 2 analysis(Larkin M. A et al (2007) Bioinformatics 23(21): 2947-2948) for multiplesequence alignment. The evolutionary history is then inferred by theMaximum Likelihood method based on the JTT matrix-based model. The treewith the highest log likelihood is obtained, exported in Newick format,and further processed to extract the sequence IDs in the same order asthey appeared in the tree. A few clades representing sub-families can bemanually identified for each insecticidal protein family.

2) Protein Sequence Motifs Finding

Protein sequences are re-ordered according to the phylogenetic treebuilt previously, and fed to the MOTIF analysis tool MEME (Multiple EMfor MOTIF Elicitation) (Bailey T. L., and Elkan C., Proceedings of theSecond International Conference on Intelligent Systems for MolecularBiology, pp. 28-36, AAAI Press, Menlo Park, Calif., 1994) foridentification of key sequence motifs. MEME is setup as follows: Minimumnumber of sites 2, Minimum motif width 5, and Maximum number of motifs30. Sequence motifs unique to each sub-family were identified by visualobservation. The distribution of MOTIFs across the entire gene familycould be visualized in HTML webpage. The MOTIFs are numbered relative tothe ranking of the E-value for each MOTIF.

3) Secondary Structure Prediction

PSIPRED, top ranked secondary structure prediction method (Jones D T.(1999) J. Mol. Biol. 292: 195-202), can be used for protein secondarystructure prediction. The tool provides accurate structure predictionusing two feed-forward neural networks based on the PSI-BLAST output.The PSI-BLAST database is created by removing low-complexity,transmembrane, and coiled-coil regions in Uniref100. The PSIPRED resultscontain the predicted secondary structures (Alpha helix: H, Beta strand:E, and Coil: C) and the corresponding confidence scores for each aminoacid in a given protein sequence.

4) Alignment of Protein Sequences and Secondary Structures

A script can be developed to generate gapped secondary structurealignment according to the multiple protein sequence alignment from step1 for all proteins. All aligned protein sequences and structures areconcatenated into a single FASTA file, and then imported into MEGA forvisualization and identification of conserved structures.

In some embodiments the IPD090 polypeptide has a modified physicalproperty. As used herein, the term “physical property” refers to anyparameter suitable for describing the physical-chemical characteristicsof a protein. As used herein, “physical property of interest” and“property of interest” are used interchangeably to refer to physicalproperties of proteins that are being investigated and/or modified.Examples of physical properties include, but are not limited to, netsurface charge and charge distribution on the protein surface, nethydrophobicity and hydrophobic residue distribution on the proteinsurface, surface charge density, surface hydrophobicity density, totalcount of surface ionizable groups, surface tension, protein size and itsdistribution in solution, melting temperature, heat capacity, and secondvirial coefficient. Examples of physical properties also include, IPD090polypeptide having increased expression, increased solubility, decreasedphytotoxicity, and digestibility of proteolytic fragments in an insectgut. Models for digestion by simulated gastric fluids are known to oneskilled in the art (Fuchs, R. L. and J. D. Astwood. Food Technology 50:83-88, 1996; Astwood, J. D., et al Nature Biotechnology 14: 1269-1273,1996; Fu T J et al J. Agric Food Chem. 50: 7154-7160, 2002).

In some embodiments variants include polypeptides that differ in aminoacid sequence due to mutagenesis. Variant proteins encompassed by thedisclosure are biologically active, that is they continue to possess thedesired biological activity (i.e. pesticidal activity) of the nativeprotein. In some embodiment the variant will have at least about 10%, atleast about 30%, at least about 50%, at least about 70%, at least about80% or more of the insecticidal activity of the native protein. In someembodiments, the variants may have improved activity over the nativeprotein.

Bacterial genes quite often possess multiple methionine initiationcodons in proximity to the start of the open reading frame. Often,translation initiation at one or more of these start codons will lead togeneration of a functional protein. These start codons can include ATGcodons. However, bacteria such as Bacillus sp. also recognize the codonGTG as a start codon, and proteins that initiate translation at GTGcodons contain a methionine at the first amino acid. On rare occasions,translation in bacterial systems can initiate at a TTG codon, though inthis event the TTG encodes a methionine. Furthermore, it is not oftendetermined a priori which of these codons are used naturally in thebacterium. Thus, it is understood that use of one of the alternatemethionine codons may also lead to generation of pesticidal proteins.These pesticidal proteins are encompassed in the present disclosure andmay be used in the methods of the present disclosure. It will beunderstood that, when expressed in plants, it will be necessary to alterthe alternate start codon to ATG for proper translation.

In some embodiments the IPD090 polypeptide comprises the amino acidsequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379 orSEQ ID NO: 384.

In some embodiments the IPD090 polypeptide comprises the amino acidsequence of SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO:117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO:126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO:135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO:144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO:153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO:162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO:171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO:180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO:189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO:198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO:278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO:287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO:296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO:305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO:314, SEQ ID NO: 315, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 318, SEQID NO: 319, SEQ ID NO: 320, SEQ ID NO: 321, SEQ ID NO: 322, SEQ ID NO:323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, SEQID NO: 328, SEQ ID NO: 329, SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO:332, SEQ ID NO: 333, SEQ ID NO: 334, SEQ ID NO: 335, SEQ ID NO: 336, SEQID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO:341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 377, SEQID NO: 379 or SEQ ID NO: 384.

In some embodiments, chimeric polypeptides are provided comprisingregions of at least two different IPD090 polypeptides of the disclosure.

In some embodiments, chimeric polypeptides are provided comprisingregions of at least two different IPD090 polypeptides selected from SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO:118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO:127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO:136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO:145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO:154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO:163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO:172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO:181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO:190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO:199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 274, SEQID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO:279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO:288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO:297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO:306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO:315, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, SEQID NO: 320, SEQ ID NO: 321, SEQ ID NO: 322, SEQ ID NO: 323, SEQ ID NO:324, SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, SEQ ID NO: 328, SEQID NO: 329, SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO:333, SEQ ID NO: 334, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, SEQID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO:342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 377, SEQ ID NO: 379, andSEQ ID NO: 384.

In some embodiments, chimeric IPD090 polypeptide are provided comprisingan N-terminal Region of a first IPD090 polypeptide of the disclosureoperably fused to a C-terminal Region of a second IPD090 polypeptide ofthe disclosure.

In some embodiments, chimeric IPD090 polypeptide are provided comprisingan N-terminal Region of a first IPD090 polypeptide operably fused to aC-terminal Region of a second IPD090 polypeptide, where the first andsecond IPD090 polypeptide is selected from SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 114, SEQ ID NO:115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO:133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO:142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO:151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO:160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO:169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO:178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO:187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO:196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQID NO: 201, SEQ ID NO: 202, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO:276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO:285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO:294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO:303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO:312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID NO: 316, SEQID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, SEQ ID NO:321, SEQ ID NO: 322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQID NO: 326, SEQ ID NO: 327, SEQ ID NO: 328, SEQ ID NO: 329, SEQ ID NO:330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, SEQID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO:339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQID NO: 344, SEQ ID NO: 377, SEQ ID NO: 379, and SEQ ID NO: 384.

In some embodiments the chimeric IPD090 polypeptide comprises: a) anN-terminal Region having at least 90% sequence identity to the aminoacid residues corresponding to amino acids 1 to about 144, amino acids 1to about 239, amino acids 1 to about 296, amino acids 1 to about 348,amino acids 1 to about 382, amino acids 1 to about 422, amino acids 1 toabout 442 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal Regionhaving at least 90% sequence identity to the amino acid residuescorresponding to amino acids of about 146 to about 483, amino acids ofabout 241 to about 483, amino acids of about 297 to about 483, aminoacids of about 349 to about 483, amino acids of about 383 to about 483,amino acids of about 423 to about 483 or amino acids of about 443 toabout 483 of SEQ ID NO: 6.

In some embodiments the chimeric IPD090 polypeptide comprises; a) anN-terminal Region having at least 90% sequence identity to the aminoacid residues corresponding to amino acids 1 to about 144 of SEQ ID NO:2 or SEQ ID NO: 4; and b) a C-terminal Region having at least 90%sequence identity to the amino acid residues corresponding to aminoacids of about 146 to 483 of SEQ ID NO: 6.

In some embodiments the chimeric IPD090 polypeptide comprises; a) anN-terminal Region having at least 90% sequence identity to the aminoacid residues corresponding to amino acids 1 to about 239 of SEQ ID NO:2 or SEQ ID NO: 4; and b) a C-terminal Region having at least 90%sequence identity to the amino acid residues corresponding to aminoacids of about 241 to 483 of SEQ ID NO: 6.

In some embodiments the chimeric IPD090 polypeptide comprises; a) anN-terminal Region having at least 90% sequence identity to the aminoacid residues corresponding to amino acids 1 to about 296 of SEQ ID NO:2 or SEQ ID NO: 4; and b) a C-terminal Region having at least 90%sequence identity to the amino acid residues corresponding to aminoacids of about 297 to about 483 of SEQ ID NO: 6.

In some embodiments the chimeric IPD090 polypeptide comprises; a) anN-terminal Region having at least 90% sequence identity to the aminoacid residues corresponding to amino acids 1 to about 348 of SEQ ID NO:2 or SEQ ID NO: 4; and b) a C-terminal Region having at least 90%sequence identity to the amino acid residues corresponding to aminoacids of about 349 to 483 of SEQ ID NO: 6.

In some embodiments the chimeric IPD090 polypeptide comprises; a) anN-terminal Region having at least 90% sequence identity to the aminoacid residues corresponding to amino acids 1 to about 382 of SEQ ID NO:2 or SEQ ID NO: 4; and b) a C-terminal Region having at least 90%sequence identity to the amino acid residues corresponding to aminoacids of about 383 to 483 of SEQ ID NO: 6.

In some embodiments the chimeric IPD090 polypeptide comprises; a) anN-terminal Region having at least 90% sequence identity to the aminoacid residues corresponding to amino acids 1 to about 422 of SEQ ID NO:2 or SEQ ID NO: 4; and b) a C-terminal Region having at least 90%sequence identity to the amino acid residues corresponding to aminoacids about 423 to 483 of SEQ ID NO: 6.

In some embodiments the chimeric IPD090 polypeptide comprises; a) anN-terminal Region having at least 90% sequence identity to the aminoacid residues corresponding to amino acids 1 to about 442 of SEQ ID NO:2 or SEQ ID NO: 4; and b) a C-terminal Region having at least 90%sequence identity to the amino acid residues corresponding to aminoacids about 443 to 483 of SEQ ID NO: 6.

In some embodiments the chimeric IPD090 polypeptide comprises; a) anN-terminal Region comprising amino acids 1 to about 144, amino acids 1to about 239, amino acids 1 to about 296, amino acids 1 to about 348,amino acids 1 to about 382, amino acids 1 to about 422, amino acids 1 toabout 442 of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6; and b) aC-terminal Region comprising the amino acids of about 146 to about 483,amino acids of about 241 to about 483, amino acids of about 297 to about483, amino acids of about 349 to about 483, amino acids of about 383 toabout 483, amino acids of about 423 to about 483 or amino acids of about443 to about 483 of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.

In some embodiments the chimeric IPD090 polypeptide comprises; a) anN-terminal Region comprising amino acids 1 to about 144 of SEQ ID NO: 2or SEQ ID NO: 4; and b) a C-terminal Region comprising amino acids ofabout 146 to 483 of SEQ ID NO: 6.

In some embodiments the chimeric IPD090 polypeptide comprises; a) anN-terminal Region comprising amino acids 1 to about 239 of SEQ ID NO: 2or SEQ ID NO: 4; and b) a C-terminal Region comprising amino acids ofabout 241 to 483 of SEQ ID NO: 6.

In some embodiments the chimeric IPD090 polypeptide comprises; a) anN-terminal Region comprising amino acids 1 to about 296 of SEQ ID NO: 2or SEQ ID NO: 4; and b) a C-terminal Region comprising amino acids ofabout 297 to about 483 of SEQ ID NO: 6.

In some embodiments the chimeric IPD090 polypeptide comprises; a) anN-terminal Region comprises amino acids 1 to about 348 of SEQ ID NO: 2or SEQ ID NO: 4; and b) a C-terminal Region comprising amino acids ofabout 349 to 483 of SEQ ID NO: 6.

In some embodiments the chimeric IPD090 polypeptide comprises; a) anN-terminal Region comprising amino acids 1 to about 382 of SEQ ID NO: 2or SEQ ID NO: 4; and b) a C-terminal Region comprising amino acids ofabout 383 to 483 of SEQ ID NO: 6.

In some embodiments the chimeric IPD090 polypeptide comprises; a) anN-terminal Region comprising amino acids 1 to about 422 of SEQ ID NO: 2or SEQ ID NO: 4; and b) a C-terminal Region comprising amino acids about423 to 483 of SEQ ID NO: 6.

In some embodiments the chimeric IPD090 polypeptide comprises; a) anN-terminal Region comprising amino acids 1 to about 442 of SEQ ID NO: 2or SEQ ID NO: 4; and b) a C-terminal Region comprising amino acids about443 to 483 of SEQ ID NO: 6.

In other embodiments the IPD090 polypeptide may be expressed as aprecursor protein with an intervening sequence that catalyzesmulti-step, post translational protein splicing. Protein splicinginvolves the excision of an intervening sequence from a polypeptide withthe concomitant joining of the flanking sequences to yield a newpolypeptide (Chong, et al., (1996) J. Biol. Chem., 271:22159-22168).This intervening sequence or protein splicing element, referred to asinteins, which catalyze their own excision through three coordinatedreactions at the N-terminal and C-terminal splice junctions: an acylrearrangement of the N-terminal cysteine or serine; a transesterficationreaction between the two termini to form a branched ester or thioesterintermediate and peptide bond cleavage coupled to cyclization of theintein C-terminal asparagine to free the intein (Evans, et al., (2000)J. Biol. Chem., 275:9091-9094. The elucidation of the mechanism ofprotein splicing has led to a number of intein-based applications (Comb,et al., U.S. Pat. No. 5,496,714; Comb, et al., U.S. Pat. No. 5,834,247;Camarero and Muir, (1999) J. Amer. Chem. Soc. 121:5597-5598; Chong, etal., (1997) Gene 192:271-281, Chong, et al., (1998) Nucleic Acids Res.26:5109-5115; Chong, et al., (1998) J. Biol. Chem. 273:10567-10577;Cotton, et al., (1999) J. Am. Chem. Soc. 121:1100-1101; Evans, et al.,(1999) J. Biol. Chem. 274:18359-18363; Evans, et al., (1999) J. Biol.Chem. 274:3923-3926; Evans, et al., (1998) Protein Sci. 7:2256-2264;Evans, et al., (2000) J. Biol. Chem. 275:9091-9094; Iwai and Pluckthun,(1999) FEBS Lett. 459:166-172; Mathys, et al., (1999) Gene 231:1-13;Mills, et al., (1998) Proc. Natl. Acad. Sci. USA 95:3543-3548; Muir, etal., (1998) Proc. Natl. Acad. Sci. USA 95:6705-6710; Otomo, et al.,(1999) Biochemistry 38:16040-16044; Otomo, et al., (1999) J. Biolmol.NMR 14:105-114; Scott, et al., (1999) Proc. Natl. Acad. Sci. USA96:13638-13643; Severinov and Muir, (1998) J. Biol. Chem.273:16205-16209; Shingledecker, et al., (1998) Gene 207:187-195;Southworth, et al., (1998) EMBO J. 17:918-926; Southworth, et al.,(1999) Biotechniques 27:110-120; Wood, et al., (1999) Nat. Biotechnol.17:889-892; Wu, et al., (1998a) Proc. Natl. Acad. Sci. USA 95:9226-9231;Wu, et al., (1998b) Biochim Biophys Acta 1387:422-432; Xu, et al.,(1999) Proc. Natl. Acad. Sci. USA 96:388-393; Yamazaki, et al., (1998)J. Am. Chem. Soc., 120:5591-5592). For the application of inteins inplant transgenes, see, Yang, et al., (Transgene Res 15:583-593 (2006))and Evans, et al., (Annu. Rev. Plant Biol. 56:375-392 (2005)).

In another embodiment the IPD090 polypeptide may be encoded by twoseparate genes where the intein of the precursor protein comes from thetwo genes, referred to as a split-intein, and the two portions of theprecursor are joined by a peptide bond formation. This peptide bondformation is accomplished by intein-mediated trans-splicing. For thispurpose, a first and a second expression cassette comprising the twoseparate genes further code for inteins capable of mediating proteintrans-splicing. By trans-splicing, the proteins and polypeptides encodedby the first and second fragments may be linked by peptide bondformation. Trans-splicing inteins may be selected from the nucleolar andorganellar genomes of different organisms including eukaryotes,archaebacteria and eubacteria. Inteins that may be used for are listedat neb.com/neb/inteins.html, which can be accessed on the world-wide webusing the “www” prefix). The nucleotide sequence coding for an inteinmay be split into a 5′ and a 3′ part that code for the 5′ and the 3′part of the intein, respectively. Sequence portions not necessary forintein splicing (e.g. homing endonuclease domain) may be deleted. Theintein coding sequence is split such that the 5′ and the 3′ parts arecapable of trans-splicing. For selecting a suitable splitting site ofthe intein coding sequence, the considerations published by Southworth,et al., (1998) EMBO J. 17:918-926 may be followed. In constructing thefirst and the second expression cassette, the 5′ intein coding sequenceis linked to the 3′ end of the first fragment coding for the N-terminalpart of the IPD090 polypeptide and the 3′ intein coding sequence islinked to the 5′ end of the second fragment coding for the C-terminalpart of the IPD090 polypeptide.

In general, the trans-splicing partners can be designed using any splitintein, including any naturally-occurring or artificially-split splitintein. Several naturally-occurring split inteins are known, forexample: the split intein of the DnaE gene of Synechocystis sp. PCC6803(see, Wu, et al., (1998) Proc Natl Acad Sci USA. 95(16):9226-31 andEvans, et al., (2000) J Biol Chem. 275(13):9091-4 and of the DnaE genefrom Nostoc punctiforme (see, Iwai, et al., (2006) FEBS Lett.580(7):1853-8). Non-split inteins have been artificially split in thelaboratory to create new split inteins, for example: the artificiallysplit Ssp DnaB intein (see, Wu, et al., (1998) Biochim Biophys Acta.1387:422-32) and split Sce VMA intein (see, Brenzel, et al., (2006)Biochemistry. 45(6):1571-8) and an artificially split fungal mini-intein(see, Elleuche, et al., (2007) Biochem Biophys Res Commun.355(3):830-4). There are also intein databases available that catalogueknown inteins (see for example the online-database available at:bioinformatics.weizmann.ac.il/^(˜)pietro/inteins/Inteinstable.html,which can be accessed on the world-wide web using the “www” prefix).

Naturally-occurring non-split inteins may have endonuclease or otherenzymatic activities that can typically be removed when designing anartificially-split split intein. Such mini-inteins or minimized splitinteins are well known in the art and are typically less than 200 aminoacid residues long (see, Wu, et al., (1998) Biochim Biophys Acta.1387:422-32). Suitable split inteins may have other purificationenabling polypeptide elements added to their structure, provided thatsuch elements do not inhibit the splicing of the split intein or areadded in a manner that allows them to be removed prior to splicing.Protein splicing has been reported using proteins that comprisebacterial intein-like (BIL) domains (see, Amitai, et al., (2003) MolMicrobiol. 47:61-73) and hedgehog (Hog) auto-processing domains (thelatter is combined with inteins when referred to as the Hog/inteinsuperfamily or HINT family (see, Dassa, et al., (2004) J Biol Chem.279:32001-7) and domains such as these may also be used to prepareartificially-split inteins. In particular, non-splicing members of suchfamilies may be modified by molecular biology methodologies to introduceor restore splicing activity in such related species. Recent studiesdemonstrate that splicing can be observed when a N-terminal split inteincomponent is allowed to react with a C-terminal split intein componentnot found in nature to be its “partner”; for example, splicing has beenobserved utilizing partners that have as little as 30 to 50% homologywith the “natural” splicing partner (see, Dassa, et al., (2007)Biochemistry. 46(1):322-30). Other such mixtures of disparate splitintein partners have been shown to be unreactive one with another (see,Brenzel, et al., (2006) Biochemistry. 45(6):1571-8). However, it iswithin the ability of a person skilled in the relevant art to determinewhether a particular pair of polypeptides is able to associate with eachother to provide a functional intein, using routine methods and withoutthe exercise of inventive skill.

In some embodiments the IPD090 polypeptide is a circular permutedvariant. In certain embodiments the IPD090 polypeptide is a circularpermuted variant of the polypeptide of, SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 379 or SEQ ID NO: 384, or variant thereof having anamino acid substitution, deletion, addition or combinations thereof. Thedevelopment of recombinant DNA methods has made it possible to study theeffects of sequence transposition on protein folding, structure andfunction. The approach used in creating new sequences resembles that ofnaturally occurring pairs of proteins that are related by linearreorganization of their amino acid sequences (Cunningham, et al., (1979)Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222; Teather and Erfle, (1990) J.Bacteriol. 172:3837-3841; Schimming, et al., (1992) Eur. J. Biochem.204:13-19; Yamiuchi and Minamikawa, (1991) FEBS Lett. 260:127-130;MacGregor, et al., (1996) FEBS Lett. 378:263-266). The first in vitroapplication of this type of rearrangement to proteins was described byGoldenberg and Creighton (J. Mol. Biol. 165:407-413, 1983). In creatinga circular permuted variant, a new N-terminus is selected at an internalsite (breakpoint) of the original sequence, the new sequence having thesame order of amino acids as the original from the breakpoint until itreaches an amino acid that is at or near the original C-terminus. Atthis point the new sequence is joined, either directly or through anadditional portion of sequence (linker), to an amino acid that is at ornear the original N-terminus and the new sequence continues with thesame sequence as the original until it reaches a point that is at ornear the amino acid that was N-terminal to the breakpoint site of theoriginal sequence, this residue forming the new C-terminus of the chain.The length of the amino acid sequence of the linker can be selectedempirically or with guidance from structural information or by using acombination of the two approaches. When no structural information isavailable, a small series of linkers can be prepared for testing using adesign whose length is varied in order to span a range from 0 to 50 Åand whose sequence is chosen in order to be consistent with surfaceexposure (hydrophilicity, Hopp and Woods, (1983) Mol. Immunol.20:483-489; Kyte and Doolittle, (1982) J. Mol. Biol. 157:105-132;solvent exposed surface area, Lee and Richards, (1971) J. Mol. Biol.55:379-400) and the ability to adopt the necessary conformation withoutderanging the configuration of the pesticidal polypeptide(conformationally flexible; Karplus and Schulz, (1985)Naturwissenschaften 72:212-213). Assuming an average of translation of2.0 to 3.8 Å per residue, this would mean the length to test would bebetween 0 to 30 residues, with 0 to 15 residues being the preferredrange. Exemplary of such an empirical series would be to constructlinkers using a cassette sequence such as Gly-Gly-Gly-Ser repeated ntimes, where n is 1, 2, 3 or 4. Those skilled in the art will recognizethat there are many such sequences that vary in length or compositionthat can serve as linkers with the primary consideration being that theybe neither excessively long nor short (cf., Sandhu, (1992) Critical Rev.Biotech. 12:437-462); if they are too long, entropy effects will likelydestabilize the three-dimensional fold, and may also make foldingkinetically impractical, and if they are too short, they will likelydestabilize the molecule because of torsional or steric strain. Thoseskilled in the analysis of protein structural information will recognizethat using the distance between the chain ends, defined as the distancebetween the c-alpha carbons, can be used to define the length of thesequence to be used or at least to limit the number of possibilitiesthat must be tested in an empirical selection of linkers. They will alsorecognize that it is sometimes the case that the positions of the endsof the polypeptide chain are ill-defined in structural models derivedfrom x-ray diffraction or nuclear magnetic resonance spectroscopy data,and that when true, this situation will therefore need to be taken intoaccount in order to properly estimate the length of the linker required.From those residues whose positions are well defined are selected tworesidues that are close in sequence to the chain ends, and the distancebetween their c-alpha carbons is used to calculate an approximate lengthfor a linker between them. Using the calculated length as a guide,linkers with a range of number of residues (calculated using 2 to 3.8 Åper residue) are then selected. These linkers may be composed of theoriginal sequence, shortened or lengthened as necessary, and whenlengthened the additional residues may be chosen to be flexible andhydrophilic as described above; or optionally the original sequence maybe substituted for using a series of linkers, one example being theGly-Gly-Gly-Ser cassette approach mentioned above; or optionally acombination of the original sequence and new sequence having theappropriate total length may be used. Sequences of pesticidalpolypeptides capable of folding to biologically active states can beprepared by appropriate selection of the beginning (amino terminus) andending (carboxyl terminus) positions from within the originalpolypeptide chain while using the linker sequence as described above.Amino and carboxyl termini are selected from within a common stretch ofsequence, referred to as a breakpoint region, using the guidelinesdescribed below. A novel amino acid sequence is thus generated byselecting amino and carboxyl termini from within the same breakpointregion. In many cases the selection of the new termini will be such thatthe original position of the carboxyl terminus immediately preceded thatof the amino terminus. However, those skilled in the art will recognizethat selections of termini anywhere within the region may function, andthat these will effectively lead to either deletions or additions to theamino or carboxyl portions of the new sequence. It is a central tenet ofmolecular biology that the primary amino acid sequence of a proteindictates folding to the three-dimensional structure necessary forexpression of its biological function. Methods are known to thoseskilled in the art to obtain and interpret three-dimensional structuralinformation using x-ray diffraction of single protein Crystals ornuclear magnetic resonance spectroscopy of protein solutions. Examplesof structural information that are relevant to the identification ofbreakpoint regions include the location and type of protein secondarystructure (alpha and 3-10 helices, parallel and anti-parallel betasheets, chain reversals and turns, and loops; Kabsch and Sander, (1983)Biopolymers 22:2577-2637; the degree of solvent exposure of amino acidresidues, the extent and type of interactions of residues with oneanother (Chothia, (1984) Ann. Rev. Biochem. 53:537-572) and the staticand dynamic distribution of conformations along the polypeptide chain(Alber and Mathews, (1987) Methods Enzymol. 154:511-533). In some cases,additional information is known about solvent exposure of residues; oneexample is a site of post-translational attachment of carbohydrate whichis necessarily on the surface of the protein. When experimentalstructural information is not available or is not feasible to obtain,methods are also available to analyze the primary amino acid sequence inorder to make predictions of protein tertiary and secondary structure,solvent accessibility and the occurrence of turns and loops. Biochemicalmethods are also sometimes applicable for empirically determiningsurface exposure when direct structural methods are not feasible; forexample, using the identification of sites of chain scission followinglimited proteolysis in order to infer surface exposure (Gentile andSalvatore, (1993) Eur. J. Biochem. 218:603-621). Thus using either theexperimentally derived structural information or predictive methods(e.g., Srinivisan and Rose, (1995) Proteins: Struct., Funct. & Genetics22:81-99) the parental amino acid sequence is inspected to classifyregions according to whether or not they are integral to the maintenanceof secondary and tertiary structure. The occurrence of sequences withinregions that are known to be involved in periodic secondary structure(alpha and 3-10 helices, parallel and anti-parallel beta sheets) areregions that should be avoided. Similarly, regions of amino acidsequence that are observed or predicted to have a low degree of solventexposure are more likely to be part of the so-called hydrophobic core ofthe protein and should also be avoided for selection of amino andcarboxyl termini. In contrast, those regions that are known or predictedto be in surface turns or loops, and especially those regions that areknown not to be required for biological activity, are the preferredsites for location of the extremes of the polypeptide chain. Continuousstretches of amino acid sequence that are preferred based on the abovecriteria are referred to as a breakpoint region. Polynucleotidesencoding circular permuted IPD090 polypeptides with newN-terminus/C-terminus which contain a linker region separating theoriginal C-terminus and N-terminus can be made essentially following themethod described in Mullins, et al., (1994) J. Am. Chem. Soc.116:5529-5533. Multiple steps of polymerase chain reaction (PCR)amplifications are used to rearrange the DNA sequence encoding theprimary amino acid sequence of the protein. Polynucleotides encodingcircular permuted IPD090 polypeptides with new N-terminus/C-terminuswhich contain a linker region separating the original C-terminus andN-terminus can be made based on the tandem-duplication method describedin Horlick, et al., (1992) Protein Eng. 5:427-431. Polymerase chainreaction (PCR) amplification of the new N-terminus/C-terminus genes isperformed using a tandemly duplicated template DNA.

In another embodiment fusion proteins are provided that include withinits amino acid sequence an amino acid sequence comprising an IPD090polypeptide or chimeric IPD090 polypeptide of the disclosure. Methodsfor design and construction of fusion proteins (and polynucleotidesencoding same) are known to those of skill in the art. Polynucleotidesencoding an IPD090 polypeptide may be fused to signal sequences whichwill direct the localization of the IPD090 polypeptide to particularcompartments of a prokaryotic or eukaryotic cell and/or direct thesecretion of the IPD090 polypeptide of the embodiments from aprokaryotic or eukaryotic cell. For example, in E. coli, one may wish todirect the expression of the protein to the periplasmic space. Examplesof signal sequences or proteins (or fragments thereof) to which theIPD090 polypeptide may be fused in order to direct the expression of thepolypeptide to the periplasmic space of bacteria include, but are notlimited to, the pelB signal sequence, the maltose binding protein (MBP)signal sequence, MBP, the ompA signal sequence, the signal sequence ofthe periplasmic E. coli heat-labile enterotoxin B-subunit and the signalsequence of alkaline phosphatase. Several vectors are commerciallyavailable for the construction of fusion proteins which will direct thelocalization of a protein, such as the pMAL series of vectors(particularly the pMAL-p series) available from New England Biolabs. Ina specific embodiment, the IPD090 polypeptide may be fused to the pelBpectate lyase signal sequence to increase the efficiency of expressionand purification of such polypeptides in Gram-negative bacteria (see,U.S. Pat. Nos. 5,576,195 and 5,846,818). Plant plastid transitpeptide/polypeptide fusions are well known in the art. Apoplast transitpeptides such as rice or barley alpha-amylase secretion signal are alsowell known in the art. The plastid transit peptide is generally fusedN-terminal to the polypeptide to be targeted (e.g., the fusion partner).In one embodiment, the fusion protein consists essentially of theplastid transit peptide and the IPD090 polypeptide to be targeted. Inanother embodiment, the fusion protein comprises the plastid transitpeptide and the polypeptide to be targeted. In such embodiments, theplastid transit peptide is preferably at the N-terminus of the fusionprotein. However, additional amino acid residues may be N-terminal tothe plastid transit peptide providing that the fusion protein is atleast partially targeted to a plastid. In a specific embodiment, theplastid transit peptide is in the N-terminal half, N-terminal third orN-terminal quarter of the fusion protein. Most or all of the plastidtransit peptide is generally cleaved from the fusion protein uponinsertion into the plastid. The position of cleavage may vary slightlybetween plant species, at different plant developmental stages, as aresult of specific intercellular conditions or the particularcombination of transit peptide/fusion partner used. In one embodiment,the plastid transit peptide cleavage is homogenous such that thecleavage site is identical in a population of fusion proteins. Inanother embodiment, the plastid transit peptide is not homogenous, suchthat the cleavage site varies by 1-10 amino acids in a population offusion proteins. The plastid transit peptide can be recombinantly fusedto a second protein in one of several ways. For example, a restrictionendonuclease recognition site can be introduced into the nucleotidesequence of the transit peptide at a position corresponding to itsC-terminal end and the same or a compatible site can be engineered intothe nucleotide sequence of the protein to be targeted at its N-terminalend. Care must be taken in designing these sites to ensure that thecoding sequences of the transit peptide and the second protein are kept“in frame” to allow the synthesis of the desired fusion protein. In somecases, it may be preferable to remove the initiator methionine of thesecond protein when the new restriction site is introduced. Theintroduction of restriction endonuclease recognition sites on bothparent molecules and their subsequent joining through recombinant DNAtechniques may result in the addition of one or more extra amino acidsbetween the transit peptide and the second protein. This generally doesnot affect targeting activity as long as the transit peptide cleavagesite remains accessible and the function of the second protein is notaltered by the addition of these extra amino acids at its N-terminus.Alternatively, one skilled in the art can create a precise cleavage sitebetween the transit peptide and the second protein (with or without itsinitiator methionine) using gene synthesis (Stemmer, et al., (1995) Gene164:49-53) or similar methods. In addition, the transit peptide fusioncan intentionally include amino acids downstream of the cleavage site.The amino acids at the N-terminus of the mature protein can affect theability of the transit peptide to target proteins to plastids and/or theefficiency of cleavage following protein import. This may be dependenton the protein to be targeted. See, e.g., Comai, et al., (1988) J. Biol.Chem. 263(29):15104-9. In some embodiments the IPD090 polypeptide isfused to a heterologous signal peptide or heterologous transit peptide.

In some embodiments fusion proteins are provide comprising an IPD090polypeptide or chimeric IPD090 polypeptide of the disclosure representedby a formula selected from the group consisting of:R¹-L-R², R²-L-R¹, R¹-R² or R²-R¹

wherein R¹ is an IPD090 polypeptide or chimeric IPD090 polypeptide ofthe disclosure and R² is a protein of interest. In some embodiments R¹and R² are an IPD090 polypeptide or chimeric IPD090 polypeptide of thedisclosure. The R¹ polypeptide is fused either directly or through alinker (L) segment to the R² polypeptide. The term “directly” definesfusions in which the polypeptides are joined without a peptide linker.Thus “L” represents a chemical bound or polypeptide segment to whichboth R¹ and R² are fused in frame, most commonly L is a linear peptideto which R¹ and R² are bound by amide bonds linking the carboxy terminusof R¹ to the amino terminus of L and carboxy terminus of L to the aminoterminus of R². By “fused in frame” is meant that there is notranslation termination or disruption between the reading frames of R¹and R². The linking group (L) is generally a polypeptide of between 1and 500 amino acids in length. The linkers joining the two molecules arepreferably designed to (1) allow the two molecules to fold and actindependently of each other, (2) not have a propensity for developing anordered secondary structure which could interfere with the functionaldomains of the two proteins, (3) have minimal hydrophobic or chargedcharacteristic which could interact with the functional protein domainsand (4) provide steric separation of R¹ and R² such that R¹ and R² couldinteract simultaneously with their corresponding receptors on a singlecell. Typically surface amino acids in flexible protein regions includeGly, Asn and Ser. Virtually any permutation of amino acid sequencescontaining Gly, Asn and Ser would be expected to satisfy the abovecriteria for a linker sequence. Other neutral amino acids, such as Thrand Ala, may also be used in the linker sequence. Additional amino acidsmay also be included in the linkers due to the addition of uniquerestriction sites in the linker sequence to facilitate construction ofthe fusions.

In some embodiments the linkers comprise sequences selected from thegroup of formulas: (Gly₃Ser)_(n), (Gly₄Ser)_(n), (Gly₅Ser)_(n),(Gly_(n)Ser)_(n) or (AlaGlySer)_(n) where n is an integer. One exampleof a highly-flexible linker is the (GlySer)-rich spacer region presentwithin the pIII protein of the filamentous bacteriophages, e.g.bacteriophages M13 or fd (Schaller, et al., 1975). This region providesa long, flexible spacer region between two domains of the pIII surfaceprotein. Also included are linkers in which an endopeptidase recognitionsequence is included. Such a cleavage site may be valuable to separatethe individual components of the fusion to determine if they areproperly folded and active in vitro. Examples of various endopeptidasesinclude, but are not limited to, Plasmin, Enterokinase, Kallikerin,Urokinase, Tissue Plasminogen activator, clostripain, Chymosin,Collagenase, Russell's Viper Venom Protease, Postproline cleavageenzyme, V8 protease, Thrombin and factor Xa. In some embodiments thelinker comprises the amino acids EEKKN (SEQ ID NO: 376) from themulti-gene expression vehicle (MGEV), which is cleaved by vacuolarproteases as disclosed in US Patent Application Publication Number US2007/0277263. In other embodiments, peptide linker segments from thehinge region of heavy chain immunoglobulins IgG, IgA, IgM, IgD or IgEprovide an angular relationship between the attached polypeptides.Especially useful are those hinge regions where the cysteines arereplaced with serines. Linkers of the present disclosure includesequences derived from murine IgG gamma 2b hinge region in which thecysteines have been changed to serines. The fusion proteins are notlimited by the form, size or number of linker sequences employed and theonly requirement of the linker is that functionally it does notinterfere adversely with the folding and function of the individualmolecules of the fusion.

Nucleic Acid Molecules, and Variants and Fragments Thereof

Isolated or recombinant nucleic acid molecules comprising nucleic acidsequences encoding IPD090 polypeptides or biologically active portionsthereof, as well as nucleic acid molecules sufficient for use ashybridization probes to identify nucleic acid molecules encodingproteins with regions of sequence homology are provided. As used herein,the term “nucleic acid molecule” refers to DNA molecules (e.g.,recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) andRNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

An “isolated” nucleic acid molecule (or DNA) is used herein to refer toa nucleic acid sequence (or DNA) that is no longer in its naturalenvironment, for example in vitro. A “recombinant” nucleic acid molecule(or DNA) is used herein to refer to a nucleic acid sequence (or DNA)that is in a recombinant bacterial or plant host cell. In someembodiments, an “isolated” or “recombinant” nucleic acid is free ofsequences (preferably protein encoding sequences) that naturally flankthe nucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For purposes of the disclosure, “isolated” or“recombinant” when used to refer to nucleic acid molecules excludesisolated chromosomes. For example, in various embodiments, therecombinant nucleic acid molecules encoding IPD090 polypeptides cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kbof nucleic acid sequences that naturally flank the nucleic acid moleculein genomic DNA of the cell from which the nucleic acid is derived.

In some embodiments an isolated nucleic acid molecule encoding IPD090polypeptides has one or more change in the nucleic acid sequencecompared to the native or genomic nucleic acid sequence. In someembodiments the change in the native or genomic nucleic acid sequenceincludes but is not limited to: changes in the nucleic acid sequence dueto the degeneracy of the genetic code; changes in the nucleic acidsequence due to the amino acid substitution, insertion, deletion and/oraddition compared to the native or genomic sequence; removal of one ormore intron; deletion of one or more upstream or downstream regulatoryregions; and deletion of the 5′ and/or 3′ untranslated region associatedwith the genomic nucleic acid sequence. In some embodiments the nucleicacid molecule encoding an IPD090 polypeptide is a non-genomic sequence.

A variety of polynucleotides that encode IPD090 polypeptides or relatedproteins are contemplated. Such polynucleotides are useful forproduction of IPD090 polypeptides in host cells when operably linked toa suitable promoter, transcription termination and/or polyadenylationsequences. Such polynucleotides are also useful as probes for isolatinghomologous or substantially homologous polynucleotides that encodeIPD090 polypeptides or related proteins.

Polynucleotides Encoding IPD090 Polypeptides

One source of polynucleotides that encode IPD090 polypeptides or relatedproteins is a Pseudomonas or Woodsholea bacterium which contains anIPD090 polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 378, and SEQ ID NO: 380, encoding an IPD090 polypeptide of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, and SEQ ID NO: 384,respectively. The polynucleotides of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 378 or SEQ ID NO: 380 can be used to express IPD090polypeptides in recombinant bacterial hosts that include but are notlimited to Agrobacterium, Bacillus, Escherichia, Salmonella, Pseudomonasand Rhizobium bacterial host cells. The polynucleotides are also usefulas probes for isolating homologous or substantially homologouspolynucleotides that encode IPD090 polypeptides or related proteins.Such probes can be used to identify homologous or substantiallyhomologous polynucleotides derived from Pseudomonas species.

Polynucleotides that encode IPD090 polypeptides can also be synthesizedde novo from an IPD090 polypeptide sequence. The sequence of thepolynucleotide gene can be deduced from an IPD090 polypeptide sequencethrough use of the genetic code. Computer programs such as“BackTranslate” (GCG™ Package, Acclerys, Inc. San Diego, Calif.) can beused to convert a peptide sequence to the corresponding nucleotidesequence encoding the peptide. Examples of IPD090 polypeptide sequencesthat can be used to obtain corresponding nucleotide encoding sequencesinclude, but are not limited to the IPD090 polypeptides of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, and SEQ ID NO: 384.Furthermore, synthetic IPD090 polynucleotide sequences of the disclosurecan be designed so that they will be expressed in plants.

In some embodiments the nucleic acid molecule encoding an IPD090polypeptide is a polynucleotide having the sequence set forth in SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 378 or SEQ ID NO: 380, andvariants, fragments and complements thereof. “Complement” is used hereinto refer to a nucleic acid sequence that is sufficiently complementaryto a given nucleic acid sequence such that it can hybridize to the givennucleic acid sequence to thereby form a stable duplex. “Polynucleotidesequence variants” is used herein to refer to a nucleic acid sequencethat except for the degeneracy of the genetic code encodes the samepolypeptide.

In some embodiments the nucleic acid molecule encoding the IPD090polypeptide is a non-genomic nucleic acid sequence. As used herein a“non-genomic nucleic acid sequence” or “non-genomic nucleic acidmolecule” or “non-genomic polynucleotide” refers to a nucleic acidmolecule that has one or more change in the nucleic acid sequencecompared to a native or genomic nucleic acid sequence. In someembodiments the change to a native or genomic nucleic acid moleculeincludes but is not limited to: changes in the nucleic acid sequence dueto the degeneracy of the genetic code; optimization of the nucleic acidsequence for expression in plants; changes in the nucleic acid sequenceto introduce at least one amino acid substitution, insertion, deletionand/or addition compared to the native or genomic sequence; removal ofone or more intron associated with the genomic nucleic acid sequence;insertion of one or more heterologous introns; deletion of one or moreupstream or downstream regulatory regions associated with the genomicnucleic acid sequence; insertion of one or more heterologous upstream ordownstream regulatory regions; deletion of the 5′ and/or 3′ untranslatedregion associated with the genomic nucleic acid sequence; insertion of aheterologous 5′ and/or 3′ untranslated region; and modification of apolyadenylation site. In some embodiments the non-genomic nucleic acidmolecule is a synthetic nucleic acid sequence.

In some embodiments the nucleic acid molecule encoding an IPD090polypeptide is a non-genomic polynucleotide having a nucleotide sequencehaving at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greateridentity, to the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 378 or SEQ ID NO: 380, wherein the IPD090polypeptide has insecticidal activity.

In some embodiments the nucleic acid molecule encodes an IPD090polypeptide comprising an amino acid sequence of, SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 379 or SEQ ID NO: 384 having 1, 2, 3, 4,5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95or more amino acid substitutions compared to the native amino acid atthe corresponding position of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 379 or SEQ ID NO: 384.

In some embodiments the nucleic acid molecule encodes an IPD090polypeptide variant comprising any one or more amino acid substitutionscorresponding to positions 3, 4, 8, 12, 15, 16, 21, 23, 24, 26, 28, 30,38, 46, 47, 50, 52, 55, 62, 63, 67, 68, 70, 73, 74, 75, 76, 80, 90, 91,94, 99, 100, 108, 115, 127, 129, 161, 169, 175, 177, 178, 180, 185, 207,213, 223, 240, 241, 247, 255, 266, 273, 275, 277, 278, 287, 288, 302,306, 309, 310, 311, 312, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384, 385, 386, 387, 388, 389, 391, 392, 395, 397, 400,401, 402, 405, 407, 410, 423, 425, 426, 431, 433, 434, 437, 438, 439,440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453,454, 455, 457, 458, 459, 460, 468, and 471 of SEQ ID NO: 2, in anycombination.

In some embodiments the nucleic acid molecule encodes an IPD090polypeptide variant comprising any one or more amino acid substitutionsof Table 10 or 12.

Also provided are nucleic acid molecules that encode transcriptionand/or translation products that are subsequently spliced to ultimatelyproduce functional IPD090 polypeptides. Splicing can be accomplished invitro or in vivo, and can involve cis- or trans-splicing. The substratefor splicing can be polynucleotides (e.g., RNA transcripts) orpolypeptides. An example of cis-splicing of a polynucleotide is where anintron inserted into a coding sequence is removed and the two flankingexon regions are spliced to generate an IPD090 polypeptide encodingsequence. An example of trans-splicing would be where a polynucleotideis encrypted by separating the coding sequence into two or morefragments that can be separately transcribed and then spliced to formthe full-length pesticidal encoding sequence. The use of a splicingenhancer sequence, which can be introduced into a construct, canfacilitate splicing either in cis or trans-splicing of polypeptides(U.S. Pat. Nos. 6,365,377 and 6,531,316). Thus, in some embodiments thepolynucleotides do not directly encode a full-length IPD090 polypeptide,but rather encode a fragment or fragments of an IPD090 polypeptide.These polynucleotides can be used to express a functional IPD090polypeptide through a mechanism involving splicing, where splicing canoccur at the level of polynucleotide (e.g., intron/exon) and/orpolypeptide (e.g., intein/extein). This can be useful, for example, incontrolling expression of pesticidal activity, since a functionalpesticidal polypeptide will only be expressed if all required fragmentsare expressed in an environment that permits splicing processes togenerate functional product. In another example, introduction of one ormore insertion sequences into a polynucleotide can facilitaterecombination with a low homology polynucleotide; use of an intron orintein for the insertion sequence facilitates the removal of theintervening sequence, thereby restoring function of the encoded variant.

Nucleic acid molecules that are fragments of these nucleic acidsequences encoding IPD090 polypeptides are also encompassed by theembodiments. “Fragment” as used herein refers to a portion of thenucleic acid sequence encoding an IPD090 polypeptide. A fragment of anucleic acid sequence may encode a biologically active portion of anIPD090 polypeptide or it may be a fragment that can be used as ahybridization probe or PCR primer using methods disclosed below. Nucleicacid molecules that are fragments of a nucleic acid sequence encoding anIPD090 polypeptide comprise at least about 150, 180, 210, 240, 270, 300,330 or 360, contiguous nucleotides or up to the number of nucleotidespresent in a full-length nucleic acid sequence encoding an IPD090polypeptide disclosed herein, depending upon the intended use.“Contiguous nucleotides” is used herein to refer to nucleotide residuesthat are immediately adjacent to one another. Fragments of the nucleicacid sequences of the embodiments will encode protein fragments thatretain the biological activity of the IPD090 polypeptide and, hence,retain insecticidal activity. “Retains insecticidal activity” is usedherein to refer to a polypeptide having at least about 10%, at leastabout 30%, at least about 50%, at least about 70%, 80%, 90%, 95% orhigher of the insecticidal activity of the full-length IPD090Aapolypeptide (SEQ ID NO: 2). In some embodiments, the insecticidalactivity is against a Lepidopteran species. In one embodiment, theinsecticidal activity is against a Coleopteran species. In someembodiments, the insecticidal activity is against one or more insectpests of the corn rootworm complex: western corn rootworm, Diabroticavirgifera; northern corn rootworm, D. barberi: Southern corn rootworm orspotted cucumber beetle; Diabrotica undecimpunctata howardi, and theMexican corn rootworm, D. virgifera zeae. In one embodiment, theinsecticidal activity is against a Diabrotica species.

In some embodiments the IPD090 polypeptide is encoded by a nucleic acidsequence sufficiently homologous to the nucleic acid sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 378 or SEQ ID NO: 380.“Sufficiently homologous” is used herein to refer to an amino acid ornucleic acid sequence that has at least about 50%, 55%, 60%, 65%, 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology comparedto a reference sequence using one of the alignment programs describedherein using standard parameters. One of skill in the art will recognizethat these values can be appropriately adjusted to determinecorresponding homology of proteins encoded by two nucleic acid sequencesby taking into account degeneracy, amino acid similarity, reading framepositioning, and the like. In some embodiments the sequence homology isagainst the full length sequence of the polynucleotide encoding anIPD090 polypeptide or against the full length sequence of an IPD090polypeptide.

In some embodiments the nucleic acid encodes an IPD090 polypeptidehaving at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence identity compared to SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 379 or SEQ ID NO: 384. In someembodiments the sequence identity is calculated using ClustalW algorithmin the ALIGNX® module of the Vector NTI® Program Suite (InvitrogenCorporation, Carlsbad, Calif.) with all default parameters. In someembodiments the sequence identity is across the entire length ofpolypeptide calculated using ClustalW algorithm in the ALIGNX module ofthe Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, Calif.)with all default parameters.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. In another embodiment, the comparison is across theentirety of the reference sequence (e.g., across the entirety of SEQ IDNO: 1). The percent identity between two sequences can be determinedusing techniques similar to those described below, with or withoutallowing gaps. In calculating percent identity, typically exact matchesare counted.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Needleman and Wunsch,(1970) J. Mol. Biol. 48(3):443-453, used GAP Version 10 software todetermine sequence identity or similarity using the following defaultparameters: % identity and % similarity for a nucleic acid sequenceusing GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmpiiscoring matrix; % identity or % similarity for an amino acid sequenceusing GAP weight of 8 and length weight of 2, and the BLOSUM62 scoringprogram. Equivalent programs may also be used. “Equivalent program” isused herein to refer to any sequence comparison program that, for anytwo sequences in question, generates an alignment having identicalnucleotide residue matches and an identical percent sequence identitywhen compared to the corresponding alignment generated by GAP Version10.

In some embodiments an IPD090 polynucleotide encodes an IPD090polypeptide comprising an amino acid sequence having at least about 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater identity across the entire length ofthe amino acid sequence of SEQ ID NO: 2.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising regions of at least two different IPD090polypeptides of the disclosure.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising regions of at least two different IPD090polypeptides selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116,SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ IDNO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125,SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ IDNO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134,SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ IDNO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143,SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ IDNO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152,SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ IDNO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161,SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ IDNO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170,SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ IDNO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179,SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ IDNO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188,SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ IDNO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197,SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ IDNO: 202, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277,SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ IDNO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286,SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ IDNO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295,SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ IDNO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304,SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ IDNO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313,SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID NO: 316, SEQ ID NO: 317, SEQ IDNO: 318, SEQ ID NO: 319, SEQ ID NO: 320, SEQ ID NO: 321, SEQ ID NO: 322,SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO: 326, SEQ IDNO: 327, SEQ ID NO: 328, SEQ ID NO: 329, SEQ ID NO: 330, SEQ ID NO: 331,SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, SEQ ID NO: 335, SEQ IDNO: 336, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340,SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ IDNO: 377, SEQ ID NO: 379, and SEQ ID NO: 384.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising an N-terminal Region of a first IPD090polypeptide of the disclosure operably fused to a C-terminal Region of asecond IPD090 polypeptide of the disclosure.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising an N-terminal Region of a first IPD090polypeptide operably fused to a C-terminal Region of a second IPD090polypeptide, where the IPD090 polypeptide is selected from SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 114,SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ IDNO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123,SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ IDNO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132,SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ IDNO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141,SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ IDNO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150,SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ IDNO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159,SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ IDNO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168,SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ IDNO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177,SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ IDNO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186,SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ IDNO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195,SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ IDNO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 274, SEQ ID NO: 275,SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ IDNO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284,SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ IDNO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293,SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ IDNO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302,SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ IDNO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311,SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315, SEQ IDNO: 316, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320,SEQ ID NO: 321, SEQ ID NO: 322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ IDNO: 325, SEQ ID NO: 326, SEQ ID NO: 327, SEQ ID NO: 328, SEQ ID NO: 329,SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ IDNO: 334, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO: 338,SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ IDNO: 343, SEQ ID NO: 344, SEQ ID NO: 377, SEQ ID NO: 379, and SEQ ID NO:384.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising; a) an N-terminal Region having at least 90%sequence identity to the amino acid residues corresponding to aminoacids 1 to about 144, amino acids 1 to about 239, amino acids 1 to about296, amino acids 1 to about 348, amino acids 1 to about 382, amino acids1 to about 422, amino acids 1 to about 442 of SEQ ID NO: 2 or SEQ ID NO:4; and b) a C-terminal Region having at least 90% sequence identity tothe amino acid residues corresponding to amino acids of about 146 toabout 483, amino acids of about 241 to about 483, amino acids of about297 to about 483, amino acids of about 349 to about 483, amino acids ofabout 383 to about 483, amino acids of about 423 to about 483 or aminoacids of about 443 to about 483 of SEQ ID NO: 6.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising; a) an N-terminal Region having at least 90%sequence identity to the amino acid residues corresponding to aminoacids 1 to about 144 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) aC-terminal Region having at least 90% sequence identity to the aminoacid residues corresponding to amino acids of about 146 to 483 of SEQ IDNO: 6.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising; a) an N-terminal Region having at least 90%sequence identity to the amino acid residues corresponding to aminoacids 1 to about 239 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) aC-terminal Region having at least 90% sequence identity to the aminoacid residues corresponding to amino acids of about 241 to 483 of SEQ IDNO: 6.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising; a) an N-terminal Region having at least 90%sequence identity to the amino acid residues corresponding to aminoacids 1 to about 296 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) aC-terminal Region having at least 90% sequence identity to the aminoacid residues corresponding to amino acids of about 297 to about 483 ofSEQ ID NO: 6.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising; a) an N-terminal Region having at least 90%sequence identity to the amino acid residues corresponding to aminoacids 1 to about 348 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) aC-terminal Region having at least 90% sequence identity to the aminoacid residues corresponding to amino acids of about 349 to 483 of SEQ IDNO: 6.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising; a) an N-terminal Region having at least 90%sequence identity to the amino acid residues corresponding to aminoacids 1 to about 382 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) aC-terminal Region having at least 90% sequence identity to the aminoacid residues corresponding to amino acids of about 383 to 483 of SEQ IDNO: 6.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising; a) an N-terminal Region having at least 90%sequence identity to the amino acid residues corresponding to aminoacids 1 to about 422 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) aC-terminal Region having at least 90% sequence identity to the aminoacid residues corresponding to amino acids about 423 to 483 of SEQ IDNO: 6.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising; a) an N-terminal Region having at least 90%sequence identity to the amino acid residues corresponding to aminoacids 1 to about 442 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) aC-terminal Region having at least 90% sequence identity to the aminoacid residues corresponding to amino acids about 443 to 483 of SEQ IDNO: 6.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising; a) an N-terminal Region comprising the acids 1to about 144, amino acids 1 to about 239, amino acids 1 to about 296,amino acids 1 to about 348, amino acids 1 to about 382, amino acids 1 toabout 422, amino acids 1 to about 442 of SEQ ID NO: 2, SEQ ID NO: 4 orSEQ ID NO: 6; and b) a C-terminal Region comprising the amino acids ofabout 146 to about 483, amino acids of about 241 to about 483, aminoacids of about 297 to about 483, amino acids of about 349 to about 483,amino acids of about 383 to about 483, amino acids of about 423 to about483 or amino acids of about 443 to about 483 of SEQ ID NO: 2, SEQ ID NO:4 or SEQ ID NO: 6.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising; a) an N-terminal Region comprising amino acids1 to about 144 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminalRegion comprising amino acids of about 146 to 483 of SEQ ID NO: 6.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising; a) an N-terminal Region comprising amino acids1 to about 239 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminalRegion comprising amino acids of about 241 to 483 of SEQ ID NO: 6.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising; a) an N-terminal Region comprising amino acids1 to about 296 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminalRegion comprising amino acids of about 297 to about 483 of SEQ ID NO: 6.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising; a) an N-terminal Region comprises amino acids 1to about 348 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal Regioncomprising amino acids of about 349 to 483 of SEQ ID NO: 6.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising; a) an N-terminal Region comprising amino acids1 to about 382 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminalRegion comprising amino acids of about 383 to 483 of SEQ ID NO: 6.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising; a) an N-terminal Region comprising amino acids1 to about 422 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminalRegion comprising amino acids about 423 to 483 of SEQ ID NO: 6.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising; a) an N-terminal Region comprising amino acids1 to about 442 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminalRegion comprising amino acids about 443 to 483 of SEQ ID NO: 6.

In some embodiments an IPD090 polynucleotide encodes an IPD090polypeptide comprising an amino acid sequence of, SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 379 or SEQ ID NO: 384, having 1, 2, 3,4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 85, 86, 87, 88, 89, 90 or more aminoacid substitutions compared to the native amino acid at thecorresponding position of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 379 or SEQ ID NO: 384.

In some embodiments an IPD090 polynucleotide encodes an IPD090polypeptide comprising an amino acid sequence having 1, 2, 3, 4, 5, 6,7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72 amino acid substitutions,in any combination, compared to the native amino acid at thecorresponding position of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 379 or SEQ ID NO: 384.

In some embodiments an IPD090 polynucleotide encodes an IPD090polypeptide comprising an amino acid sequence having 1, 2, 3, 4, 5, 6,7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47 or 48 amino acid substitutions, in any combination,compared to the native amino acid at the corresponding position of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379 or SEQ ID NO: 384.

In some embodiments an IPD090 polynucleotide encodes an IPD090polypeptide comprising an amino acid sequence having 1, 2, 3, 4, 5, 6,7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24amino acid substitutions, in any combination, compared to the nativeamino acid at the corresponding position of SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 379 or SEQ ID NO: 384.

In some embodiments an IPD090 polynucleotide encodes the IPD090polypeptide comprising an amino acid sequence of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 114, SEQ ID NO: 115, SEQID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO:120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO:129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO:138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO:147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO:156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO:165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO:174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO:183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO:192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO:201, SEQ ID NO: 202, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO:281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO:290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO:299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO:308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID NO: 316, SEQ ID NO:317, SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, SEQ ID NO: 321, SEQID NO: 322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO:326, SEQ ID NO: 327, SEQ ID NO: 328, SEQ ID NO: 329, SEQ ID NO: 330, SEQID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, SEQ ID NO:335, SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO:344, SEQ ID NO: 377, SEQ ID NO: 379, and SEQ ID NO: 384.

The embodiments also encompass nucleic acid molecules encoding IPD090polypeptide variants. “Variants” of the IPD090 polypeptide encodingnucleic acid sequences include those sequences that encode the IPD090polypeptides disclosed herein but that differ conservatively because ofthe degeneracy of the genetic code as well as those that aresufficiently identical as discussed above. Naturally occurring allelicvariants can be identified with the use of well-known molecular biologytechniques, such as polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleic acid sequences alsoinclude synthetically derived nucleic acid sequences that have beengenerated, for example, by using site-directed mutagenesis but whichstill encode the IPD090 polypeptides disclosed as discussed below.

The present disclosure provides isolated or recombinant polynucleotidesthat encode any of the IPD090 polypeptides disclosed herein. Thosehaving ordinary skill in the art will readily appreciate that due to thedegeneracy of the genetic code, a multitude of nucleotide sequencesencoding IPD090 polypeptides of the present disclosure exist.

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleic acid sequences thereby leading tochanges in the amino acid sequence of the encoded IPD090 polypeptides,without altering the biological activity of the proteins. Thus, variantnucleic acid molecules can be created by introducing one or morenucleotide substitutions, additions and/or deletions into thecorresponding nucleic acid sequence disclosed herein, such that one ormore amino acid substitutions, additions or deletions are introducedinto the encoded protein. Mutations can be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleic acid sequences are also encompassed bythe present disclosure.

Alternatively, variant nucleic acid sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forability to confer pesticidal activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedrecombinantly, and the activity of the protein can be determined usingstandard assay techniques.

The polynucleotides of the disclosure and fragments thereof areoptionally used as substrates for a variety of recombination andrecursive recombination reactions, in addition to standard cloningmethods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., toproduce additional pesticidal polypeptide homologues and fragmentsthereof with desired properties. A variety of such reactions are known,including those developed by the inventors and their co-workers. Methodsfor producing a variant of any nucleic acid listed herein comprisingrecursively recombining such polynucleotide with a second (or more)polynucleotide, thus forming a library of variant polynucleotides arealso embodiments of the disclosure, as are the libraries produced, thecells comprising the libraries and any recombinant polynucleotideproduced by such methods. Additionally, such methods optionally compriseselecting a variant polynucleotide from such libraries based onpesticidal activity, as is wherein such recursive recombination is donein vitro or in vivo.

A variety of diversity generating protocols, including nucleic acidrecursive recombination protocols are available and fully described inthe art. The procedures can be used separately, and/or in combination toproduce one or more variants of a nucleic acid or set of nucleic acids,as well as variants of encoded proteins. Individually and collectively,these procedures provide robust, widely applicable ways of generatingdiversified nucleic acids and sets of nucleic acids (including, e.g.,nucleic acid libraries) useful, e.g., for the engineering or rapidevolution of nucleic acids, proteins, pathways, cells and/or organismswith new and/or improved characteristics.

While distinctions and classifications are made in the course of theensuing discussion for clarity, it will be appreciated that thetechniques are often not mutually exclusive. Indeed, the various methodscan be used singly or in combination, in parallel or in series, toaccess diverse sequence variants.

The result of any of the diversity generating procedures describedherein can be the generation of one or more nucleic acids, which can beselected or screened for nucleic acids with or which confer desirableproperties or that encode proteins with or which confer desirableproperties. Following diversification by one or more of the methodsherein or otherwise available to one of skill, any nucleic acids thatare produced can be selected for a desired activity or property, e.g.pesticidal activity or, such activity at a desired pH, etc. This caninclude identifying any activity that can be detected, for example, inan automated or automatable format, by any of the assays in the art,see, e.g., discussion of screening of insecticidal activity, infra. Avariety of related (or even unrelated) properties can be evaluated, inserial or in parallel, at the discretion of the practitioner.

Descriptions of a variety of diversity generating procedures forgenerating modified nucleic acid sequences, e.g., those coding forpolypeptides having pesticidal activity or fragments thereof, are foundin the following publications and the references cited therein: Soong,et al., (2000) Nat Genet 25(4):436-439; Stemmer, et al., (1999) TumorTargeting 4:1-4; Ness, et al., (1999) Nat Biotechnol 17:893-896; Chang,et al., (1999) Nat Biotechnol 17:793-797; Minshull and Stemmer, (1999)Curr Opin Chem Biol 3:284-290; Christians, et al., (1999) Nat Biotechnol17:259-264; Crameri, et al., (1998) Nature 391:288-291; Crameri, et al.,(1997) Nat Biotechnol 15:436-438; Zhang, et al., (1997) PNAS USA94:4504-4509; Patten, et al., (1997) Curr Opin Biotechnol 8:724-733;Crameri, et al., (1996) Nat Med 2:100-103; Crameri, et al., (1996) NatBiotechnol 14:315-319; Gates, et al., (1996) J Mol Biol 255:373-386;Stemmer, (1996) “Sexual PCR and Assembly PCR” In: The Encyclopedia ofMolecular Biology. VCH Publishers, New York. pp. 447-457; Crameri andStemmer, (1995) BioTechniques 18:194-195; Stemmer, et al., (1995) Gene,164:49-53; Stemmer, (1995) Science 270: 1510; Stemmer, (1995)Bio/Technology 13:549-553; Stemmer, (1994) Nature 370:389-391 andStemmer, (1994) PNAS USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling, et al., (1997) Anal Biochem254(2):157-178; Dale, et al., (1996) Methods Mol Biol 57:369-374; Smith,(1985) Ann Rev Genet 19:423-462; Botstein and Shortle, (1985) Science229:1193-1201; Carter, (1986) Biochem J 237:1-7 and Kunkel, (1987) “Theefficiency of oligonucleotide directed mutagenesis” in Nucleic Acids &Molecular Biology (Eckstein and Lilley, eds., Springer Verlag, Berlin));mutagenesis using uracil containing templates (Kunkel, (1985) PNAS USA82:488-492; Kunkel, et al., (1987) Methods Enzymol 154:367-382 and Bass,et al., (1988) Science 242:240-245); oligonucleotide-directedmutagenesis (Zoller and Smith, (1983) Methods Enzymol 100:468-500;Zoller and Smith, (1987) Methods Enzymol 154:329-350 (1987); Zoller andSmith, (1982) Nucleic Acids Res 10:6487-6500), phosphorothioate-modifiedDNA mutagenesis (Taylor, et al., (1985) Nucl Acids Res 13:8749-8764;Taylor, et al., (1985) Nucl Acids Res 13:8765-8787 (1985); Nakamaye andEckstein, (1986) Nucl Acids Res 14:9679-9698; Sayers, et al., (1988)Nucl Acids Res 16:791-802 and Sayers, et al., (1988) Nucl Acids Res16:803-814); mutagenesis using gapped duplex DNA (Kramer, et al., (1984)Nucl Acids Res 12:9441-9456; Kramer and Fritz, (1987) Methods Enzymol154:350-367; Kramer, et al., (1988) Nucl Acids Res 16:7207 and Fritz, etal., (1988) Nucl Acids Res 16:6987-6999).

Additional suitable methods include point mismatch repair (Kramer, etal., (1984) Cell 38:879-887), mutagenesis using repair-deficient hoststrains (Carter, et al., (1985) Nucl Acids Res 13:4431-4443 and Carter,(1987) Methods in Enzymol 154:382-403), deletion mutagenesis(Eghtedarzadeh and Henikoff, (1986) Nucl Acids Res 14:5115),restriction-selection and restriction-purification (Wells, et al.,(1986) Phil Trans R Soc Lond A 317:415-423), mutagenesis by total genesynthesis (Nambiar, et al., (1984) Science 223:1299-1301; Sakamar andKhorana, (1988) Nucl Acids Res 14:6361-6372; Wells, et al., (1985) Gene34:315-323 and Grundström, et al., (1985) Nucl Acids Res 13:3305-3316),double-strand break repair (Mandecki, (1986) PNAS USA, 83:7177-7181 andArnold, (1993) Curr Opin Biotech 4:450-455). Additional details on manyof the above methods can be found in Methods Enzymol Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Additional details regarding various diversity generating methods can befound in the following US Patents, PCT Publications and Applications andEPO publications: U.S. Pat. Nos. 5,723,323, 5,763,192, 5,814,476,5,817,483, 5,824,514, 5,976,862, 5,605,793, 5,811,238, 5,830,721,5,834,252, 5,837,458, WO 1995/22625, WO 1996/33207, WO 1997/20078, WO1997/35966, WO 1999/41402, WO 1999/41383, WO 1999/41369, WO 1999/41368,EP 752008, EP 0932670, WO 1999/23107, WO 1999/21979, WO 1998/31837, WO1998/27230, WO 1998/27230, WO 2000/00632, WO 2000/09679, WO 1998/42832,WO 1999/29902, WO 1998/41653, WO 1998/41622, WO 1998/42727, WO2000/18906, WO 2000/04190, WO 2000/42561, WO 2000/42559, WO 2000/42560,WO 2001/23401 and PCT/US01/06775.

The nucleotide sequences of the embodiments can also be used to isolatecorresponding sequences from a bacterial source, including but notlimited to a Pseudomonas species. In this manner, methods such as PCR,hybridization, and the like can be used to identify such sequences basedon their sequence homology to the sequences set forth herein. Sequencesthat are selected based on their sequence identity to the entiresequences set forth herein or to fragments thereof are encompassed bythe embodiments. Such sequences include sequences that are orthologs ofthe disclosed sequences. The term “orthologs” refers to genes derivedfrom a common ancestral gene and which are found in different species asa result of speciation. Genes found in different species are consideredorthologs when their nucleotide sequences and/or their encoded proteinsequences share substantial identity as defined elsewhere herein.Functions of orthologs are often highly conserved among species.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook, et al., (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.), hereinafter “Sambrook”. See also, Innis, et al., eds.(1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

To identify potential IPD090 polypeptides from bacterium collections,the bacterial cell lysates can be screened with antibodies generatedagainst an IPD090 polypeptides and/or IPD090 polypeptides using Westernblotting and/or ELISA methods. This type of assays can be performed in ahigh throughput fashion. Positive samples can be further analyzed byvarious techniques such as antibody based protein purification andidentification. Methods of generating antibodies are well known in theart as discussed infra.

Alternatively, mass spectrometry based protein identification method canbe used to identify homologs of IPD090 polypeptides using protocols inthe literatures (Scott Patterson, (1998), 10.22, 1-24, Current Protocolin Molecular Biology published by John Wiley & Son Inc). Specifically,LC-MS/MS based protein identification method is used to associate the MSdata of given cell lysate or desired molecular weight enriched samples(excised from SDS-PAGE gel of relevant molecular weight bands to IPD090polypeptides) with sequence information of IPD090 polypeptides of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 and theirhomologs. Any match in peptide sequences indicates the potential ofhaving the homologous proteins in the samples. Additional techniques(protein purification and molecular biology) can be used to isolate theprotein and identify the sequences of the homologs.

In hybridization methods, all or part of the pesticidal nucleic acidsequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook and Russell, (2001), supra. Theso-called hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments or other oligonucleotides and may be labeledwith a detectable group such as 32P or any other detectable marker, suchas other radioisotopes, a fluorescent compound, an enzyme or an enzymeco-factor. Probes for hybridization can be made by labeling syntheticoligonucleotides based on the known IPD090 polypeptide-encoding nucleicacid sequence disclosed herein. Degenerate primers designed on the basisof conserved nucleotides or amino acid residues in the nucleic acidsequence or encoded amino acid sequence can additionally be used. Theprobe typically comprises a region of nucleic acid sequence thathybridizes under stringent conditions to at least about 12, at leastabout 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutivenucleotides of nucleic acid sequence encoding an IPD090 polypeptide ofthe disclosure or a fragment or variant thereof. Methods for thepreparation of probes for hybridization are generally known in the artand are disclosed in Sambrook and Russell, (2001), supra, hereinincorporated by reference.

For example, an entire nucleic acid sequence, encoding an IPD090polypeptide, disclosed herein or one or more portions thereof may beused as a probe capable of specifically hybridizing to correspondingnucleic acid sequences encoding IPD090 polypeptide-like sequences andmessenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique and arepreferably at least about 10 nucleotides in length or at least about 20nucleotides in length. Such probes may be used to amplify correspondingpesticidal sequences from a chosen organism by PCR. This technique maybe used to isolate additional coding sequences from a desired organismor as a diagnostic assay to determine the presence of coding sequencesin an organism. Hybridization techniques include hybridization screeningof plated DNA libraries (either plaques or colonies; see, for example,Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. “Stringent conditions” or “stringent hybridizationconditions” is used herein to refer to conditions under which a probewill hybridize to its target sequence to a detectably greater degreethan to other sequences (e.g., at least 2-fold over background).Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences that are 100%complementary to the probe can be identified (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, preferably less than 500 nucleotides inlength

Compositions

Compositions comprising at least one IPD090 polypeptide or IPD090chimeric polypeptide of the disclosure are also embraced. In oneembodiment the composition comprises an IPD090 polypeptide of thedisclosure and an agriculturally accepted carrier.

One embodiment of the disclosure relates to a composition comprising anIPD090 polypeptide of the discloser and an entomopathogenic fungalstrain selected from Metarhizium robertsii and Metarhizium anisopliae.In certain embodiments, the fungal entomopathogen comprises a spore, amicrosclerotia, or a conidia. In some embodiments, a fungalentomopathogen has insecticidal activity.

In one embodiment, the disclosure relates to a composition forincreasing resistance to a plant pest, pathogen, or insect or forincreasing plant health and/or yield comprising an IPD090 polypeptide ofthe discloser and one or more entomopathogenic fungal strains selectedfrom the group consisting of Metarhizium anisopliae 15013-1 (NRRL67073), Metarhizium robertsii 23013-3 (NRRL 67075), Metarhiziumanisopliae 3213-1 (NRRL 67074), or any combinations thereof. In anotherembodiment, the disclosure relates to a composition comprising an IPD090polypeptide of the discloser, an agriculturally accepted carrier, and afungal entomopathogen selected from the group consisting of Metarhiziumanisopliae 15013-1, Metarhizium robertsii 23013-3, Metarhiziumanisopliae 3213-1, or any combinations thereof. In a further embodiment,the fungal entomopathogen comprises a spore, conidia, or microsclerotia.In another embodiment, the disclosure relates to a compositioncomprising an IPD090 polypeptide of the discloser and one or moreentomopathogenic fungal strains selected from the group consisting ofMetarhizium anisopliae 15013-1 (NRRL 67073), Metarhizium robertsii23013-3 (NRRL 67075), Metarhizium anisopliae 3213-1 (NRRL 67074),mutants of these strains, a metabolite or combination of metabolitesproduced by a strain disclosed herein that exhibits insecticidalactivity towards a plant pest, pathogen or insect, or any combinationsthereof.

Antibodies

Antibodies to an IPD090 polypeptide of the embodiments or to variants orfragments thereof are also encompassed. The antibodies of the disclosureinclude polyclonal and monoclonal antibodies as well as fragmentsthereof which retain their ability to bind to an IPD090 polypeptidefound in the insect gut. An antibody, monoclonal antibody or fragmentthereof is said to be capable of binding a molecule if it is capable ofspecifically reacting with the molecule to thereby bind the molecule tothe antibody, monoclonal antibody or fragment thereof. The term“antibody” (Ab) or “monoclonal antibody” (Mab) is meant to includeintact molecules as well as fragments or binding regions or domainsthereof (such as, for example, Fab and F(ab).sub.2 fragments) which arecapable of binding hapten. Such fragments are typically produced byproteolytic cleavage, such as papain or pepsin. Alternatively,hapten-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry. Methods forthe preparation of the antibodies of the present disclosure aregenerally known in the art. For example, see, Antibodies, A LaboratoryManual, Ed Harlow and David Lane (eds.) Cold Spring Harbor Laboratory,N.Y. (1988), as well as the references cited therein. Standard referenceworks setting forth the general principles of immunology include: Klein,J. Immunology: The Science of Cell-Noncell Discrimination, John Wiley &Sons, N.Y. (1982); Dennett, et al., Monoclonal Antibodies, Hybridoma: ANew Dimension in Biological Analyses, Plenum Press, N.Y. (1980) andCampbell, “Monoclonal Antibody Technology,” In Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 13, Burdon, et al., (eds.),Elsevier, Amsterdam (1984). See also, U.S. Pat. Nos. 4,196,265;4,609,893; 4,713,325; 4,714,681; 4,716,111; 4,716,117 and 4,720,459.Antibodies against IPD090 polypeptides or antigen-binding portionsthereof can be produced by a variety of techniques, includingconventional monoclonal antibody methodology, for example the standardsomatic cell hybridization technique of Kohler and Milstein, (1975)Nature 256:495. Other techniques for producing monoclonal antibody canalso be employed such as viral or oncogenic transformation of Blymphocytes. An animal system for preparing hybridomas is a murinesystem. Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known. The antibodyand monoclonal antibodies of the disclosure can be prepared by utilizingan IPD090 polypeptide as antigens.

A kit for detecting the presence of an IPD090 polypeptide or detectingthe presence of a nucleotide sequence encoding an IPD090 polypeptide ina sample is provided. In one embodiment, the kit provides antibody-basedreagents for detecting the presence of an IPD090 polypeptide in a tissuesample. In another embodiment, the kit provides labeled nucleic acidprobes useful for detecting the presence of one or more polynucleotidesencoding an IPD090 polypeptide. The kit is provided along withappropriate reagents and controls for carrying out a detection method,as well as instructions for use of the kit.

Receptor Identification and Isolation

Receptors to the IPD090 polypeptide of the embodiments or to variants orfragments thereof are also encompassed. Methods for identifyingreceptors are well known in the art (see, Hofmann, et. al., (1988) Eur.J. Biochem. 173:85-91; Gill, et al., (1995) J. Biol. Chem. 27277-27282)can be employed to identify and isolate the receptor that recognizes theIPD090 polypeptide using the brush-border membrane vesicles fromsusceptible insects. In addition to the radioactive labeling methodlisted in the cited literatures, an IPD090 polypeptide can be labeledwith fluorescent dye and other common labels such as streptavidin.Brush-border membrane vesicles (BBMV) of susceptible insects such assoybean looper and stink bugs can be prepared according to the protocolslisted in the references and separated on SDS-PAGE gel and blotted onsuitable membrane. Labeled IPD090 polypeptide can be incubated withblotted membrane of BBMV and labeled IPD090 polypeptide can beidentified with the labeled reporters. Identification of protein band(s)that interact with the IPD090 polypeptide can be detected by N-terminalamino acid gas phase sequencing or mass spectrometry based proteinidentification method (Patterson, (1998) 10.22, 1-24, Current Protocolin Molecular Biology published by John Wiley & Son Inc). Once theprotein is identified, the corresponding gene can be cloned from genomicDNA or cDNA library of the susceptible insects and binding affinity canbe measured directly with the IPD090 polypeptide. Receptor function forinsecticidal activity by the IPD090 polypeptide can be verified byaccomplished by RNAi type of gene knock out method (Rajagopal, et al.,(2002) J. Biol. Chem. 277:46849-46851).

Nucleotide Constructs, Expression Cassettes and Vectors

The use of the term “nucleotide constructs” herein is not intended tolimit the embodiments to nucleotide constructs comprising DNA. Those ofordinary skill in the art will recognize that nucleotide constructsparticularly polynucleotides and oligonucleotides composed ofribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides may also be employed in the methods disclosedherein. The nucleotide constructs, nucleic acids, and nucleotidesequences of the embodiments additionally encompass all complementaryforms of such constructs, molecules, and sequences. Further, thenucleotide constructs, nucleotide molecules, and nucleotide sequences ofthe embodiments encompass all nucleotide constructs, molecules, andsequences which can be employed in the methods of the embodiments fortransforming plants including, but not limited to, those comprised ofdeoxyribonucleotides, ribonucleotides, and combinations thereof. Suchdeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues. The nucleotide constructs,nucleic acids, and nucleotide sequences of the embodiments alsoencompass all forms of nucleotide constructs including, but not limitedto, single-stranded forms, double-stranded forms, hairpins,stem-and-loop structures and the like.

A further embodiment relates to a transformed organism such as anorganism selected from plant and insect cells, bacteria, yeast,baculovirus, protozoa, nematodes and algae. The transformed organismcomprises a DNA molecule of the embodiments, an expression cassettecomprising the DNA molecule or a vector comprising the expressioncassette, which may be stably incorporated into the genome of thetransformed organism.

The sequences of the embodiments are provided in DNA constructs forexpression in the organism of interest. The construct will include 5′and 3′ regulatory sequences operably linked to a sequence of theembodiments. The term “operably linked” as used herein refers to afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand where necessary to join two protein coding regions in the samereading frame. The construct may additionally contain at least oneadditional gene to be cotransformed into the organism. Alternatively,the additional gene(s) can be provided on multiple DNA constructs.

Such a DNA construct is provided with a plurality of restriction sitesfor insertion of the IPD090 polypeptide gene sequence of the disclosureto be under the transcriptional regulation of the regulatory regions.The DNA construct may additionally contain selectable marker genes.

The DNA construct will generally include in the 5′ to 3′ direction oftranscription: a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the embodiments, and atranscriptional and translational termination region (i.e., terminationregion) functional in the organism serving as a host. Thetranscriptional initiation region (i.e., the promoter) may be native,analogous, foreign or heterologous to the host organism and/or to thesequence of the embodiments. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. The term“foreign” as used herein indicates that the promoter is not found in thenative organism into which the promoter is introduced. Where thepromoter is “foreign” or “heterologous” to the sequence of theembodiments, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked sequence of theembodiments. As used herein, a chimeric gene comprises a coding sequenceoperably linked to a transcription initiation region that isheterologous to the coding sequence. Where the promoter is a native ornatural sequence, the expression of the operably linked sequence isaltered from the wild-type expression, which results in an alteration inphenotype.

In some embodiments the DNA construct comprises a polynucleotideencoding an IPD090 polypeptide of the embodiments.

In some embodiments the DNA construct comprises a polynucleotideencoding a chimeric IPD090 polypeptide of the embodiments.

In some embodiments the DNA construct comprises a polynucleotideencoding a fusion protein comprising an IPD090 polypeptide of theembodiments.

In some embodiments the DNA construct comprises a polynucleotidecomprising a first coding sequence encoding the N-terminal Region of afirst IPD090 polypeptide of the disclosure and a second coding sequenceencoding the C-terminal Region of a second IPD090 polypeptide of thedisclosure.

In some embodiments the DNA construct comprises a polynucleotideencoding the polypeptide of SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO:387 or SEQ ID NO: 388. In some embodiments the DNA construct comprises apolynucleotide of SEQ ID NO: 381, SEQ ID NO: 382 or SEQ ID NO: 383.

In some embodiments the DNA construct may also include a transcriptionalenhancer sequence. As used herein, the term an “enhancer” refers to aDNA sequence which can stimulate promoter activity, and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Various enhancers areknown in the art including for example, introns with gene expressionenhancing properties in plants (US Patent Application Publication Number2009/0144863, the ubiquitin intron (i.e., the maize ubiquitin intron 1(see, for example, NCBI sequence S94464)), the omega enhancer or theomega prime enhancer (Gallie, et al., (1989) Molecular Biology of RNAed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990)EMBO J. 9:1685-96) and the enhancers of U.S. Pat. No. 7,803,992 may alsobe used, each of which is incorporated by reference. U.S. Pat. No.8,785,612 discloses the sugarcane bacilliform badnavirus (SCBV)transcriptional enhancer. The above list of transcriptional enhancers isnot meant to be limiting. Any appropriate transcriptional enhancer canbe used in the embodiments.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host or may be derived from another source(i.e., foreign or heterologous to the promoter, the sequence ofinterest, the plant host or any combination thereof).

Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau, et al., (1991) Mol. Gen.Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al.,(1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al.,(1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., (1987) NucleicAcid Res. 15:9627-9639. Other useful transcription terminators forexpression of transgenes in plants include the transcription terminatorsMYB2, KTI1, PIP1, EF1A2, and MTH1 of U.S. Pat. No. 8,741,634.

Where appropriate, a nucleic acid may be optimized for increasedexpression in the host organism. Thus, where the host organism is aplant, the synthetic nucleic acids can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred usage. For example, although nucleic acid sequences ofthe embodiments may be expressed in both monocotyledonous anddicotyledonous plant species, sequences can be modified to account forthe specific preferences and GC content preferences of monocotyledons ordicotyledons as these preferences have been shown to differ (Murray etal. (1989) Nucleic Acids Res. 17:477-498). Thus, the maize-preferred fora particular amino acid may be derived from known gene sequences frommaize. Maize usage for 28 genes from maize plants is listed in Table 4of Murray, et al., supra. Methods are available in the art forsynthesizing plant-preferred genes. See, for example, Murray, et al.,(1989) Nucleic Acids Res. 17:477-498, and Liu H et al. Mol Bio Rep37:677-684, 2010, herein incorporated by reference. A Zea maize usagetable can be also found at kazusa.or.jp//cgi-bin/show.cgi?species=4577,which can be accessed using the www prefix.

A Glycine max usage table can be found atkazusa.or.jp//cgi-bin/show.cgi?species=3847&aa=1&style=N, which can beaccessed using the www prefix.

In some embodiments the recombinant nucleic acid molecule encoding anIPD090 polypeptide has maize optimized codons.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other well-characterized sequences that maybe deleterious to gene expression. The GC content of the sequence may beadjusted to levels average for a given cellular host, as calculated byreference to known genes expressed in the host cell. The term “hostcell” as used herein refers to a cell which contains a vector andsupports the replication and/or expression of the expression vector isintended. Host cells may be prokaryotic cells such as E. coli oreukaryotic cells such as yeast, insect, amphibian or mammalian cells ormonocotyledonous or dicotyledonous plant cells. An example of amonocotyledonous host cell is a maize host cell. When possible, thesequence is modified to avoid predicted hairpin secondary mRNAstructures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie,et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus), human immunoglobulin heavy-chain binding protein (BiP) (Macejak,et al., (1991) Nature 353:90-94); untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al.,(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie,et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York),pp. 237-256) and maize chlorotic mottle virus leader (MCMV) (Lommel, etal., (1991) Virology 81:382-385). See also, Della-Cioppa, et al., (1987)Plant Physiol. 84:965-968. Such constructs may also contain a “signalsequence” or “leader sequence” to facilitate cotranslational orpost-translational transport of the peptide to certain intracellularstructures such as the chloroplast (or other plastid), endoplasmicreticulum or Golgi apparatus.

“Signal sequence” as used herein refers to a sequence that is known orsuspected to result in cotranslational or post-translational peptidetransport across the cell membrane. In eukaryotes, this typicallyinvolves secretion into the Golgi apparatus, with some resultingglycosylation. Insecticidal toxins of bacteria are often synthesized asprotoxins, which are proteolytically activated in the gut of the targetpest (Chang, (1987) Methods Enzymol. 153:507-516). In some embodiments,the signal sequence is located in the native sequence or may be derivedfrom a sequence of the embodiments. “Leader sequence” as used hereinrefers to any sequence that when translated, results in an amino acidsequence sufficient to trigger cotranslational transport of the peptidechain to a subcellular organelle. Thus, this includes leader sequencestargeting transport and/or glycosylation by passage into the endoplasmicreticulum, passage to vacuoles, plastids including chloroplasts,mitochondria, and the like. Nuclear-encoded proteins targeted to thechloroplast thylakoid lumen compartment have a characteristic bipartitetransit peptide, composed of a stromal targeting signal peptide and alumen targeting signal peptide. The stromal targeting information is inthe amino-proximal portion of the transit peptide. The lumen targetingsignal peptide is in the carboxyl-proximal portion of the transitpeptide, and contains all the information for targeting to the lumen.Recent research in proteomics of the higher plant chloroplast hasachieved in the identification of numerous nuclear-encoded lumenproteins (Kieselbach et al. FEBS LETT 480:271-276, 2000; Peltier et al.Plant Cell 12:319-341, 2000; Bricker et al. Biochim. Biophys Acta1503:350-356, 2001), the lumen targeting signal peptide of which canpotentially be used in accordance with the present disclosure. About 80proteins from Arabidopsis, as well as homologous proteins from spinachand garden pea, are reported by Kieselbach et al., PhotosynthesisResearch, 78:249-264, 2003. In particular, Table 2 of this publication,which is incorporated into the description herewith by reference,discloses 85 proteins from the chloroplast lumen, identified by theiraccession number (see also US Patent Application Publication2009/09044298). In addition, the recently published draft version of therice genome (Goff et al, Science 296:92-100, 2002) is a suitable sourcefor lumen targeting signal peptide which may be used in accordance withthe present disclosure.

Suitable chloroplast transit peptides (CTP) are well known to oneskilled in the art also include chimeric CT's comprising but not limitedto: an N-terminal domain, a central domain or a C-terminal domain from aCTP from Oryza sativa 1-decoy-D xylose-5-Phosphate Synthase Oryzasativa-Superoxide dismutase Oryza sativa-soluble starch synthase Oryzasativa-NADP-dependent Malic acid enzyme Oryzasativa-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2 Oryzasativa-L-Ascorbate peroxidase 5 Oryza sativa-Phosphoglucan waterdikinase, Zea Mays ssRUBISCO, Zea Mays-beta-glucosidase, Zea Mays-Malatedehydrogenase, Zea Mays Thioredoxin M-type (U.S. Pat. No. 9,150,625); achloroplast transit peptide of US Patent Application Publication NumberUS20130210114.

The IPD090 polypeptide gene to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin usage between the plant nucleus and this organelle. In this manner,the nucleic acids of interest may be synthesized usingchloroplast-preferred sequences.

In preparing the expression cassette, the various DNA fragments may bemanipulated so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the embodiments.The promoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-preferred, inducible orother promoters for expression in the host organism. Suitableconstitutive promoters for use in a plant host cell include, forexample, the core promoter of the Rsyn7 promoter and other constitutivepromoters disclosed in WO 1999/43838 and U.S. Pat. No. 6,072,050; thecore CaMV 35S promoter (Odell, et al., (1985) Nature 313:810-812); riceactin (McElroy, et al., (1990) Plant Cell 2:163-171); ubiquitin(Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 andChristensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, etal., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984)EMBO J. 3:2723-2730), U.S. Pat. Nos. 8,168,859, 8,420,797; Ubiquitintranscriptional regulatory elements and transcriptional regulatoryexpression element group are disclosed in U.S. Pat. No. 9,062,316; ALSpromoter (U.S. Pat. No. 5,659,026) and the like. The Soybean ADF1constitutive promoter is disclosed in US Patent Application PublicationUS20150184174. The Soybean CCP1 constitutive promoter is disclosed in USPatent Application Publication US20150167011. Other constitutivepromoters include, for example, those discussed in U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; 5,608,142 and 6,177,611. Transcriptional initiation regionsisolated from a blueberry red ringspot virus (BRRV) are disclosed in USPatent U.S. Pat. No. 8,895,716. Transcriptional initiation regionsisolated from a cacao swollen shoot virus (CSSV) are disclosed in USPatent U.S. Pat. No. 8,962,916.

Depending on the desired outcome, it may be beneficial to express thegene from an inducible promoter. Of particular interest for regulatingthe expression of the nucleotide sequences of the embodiments in plantsare wound-inducible promoters. Such wound-inducible promoters, mayrespond to damage caused by insect feeding, and include potatoproteinase inhibitor (pin II) gene (Ryan, (1990) Ann. Rev. Phytopath.28:425-449; Duan, et al., (1996) Nature Biotechnology 14:494-498); wun1and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford, et al.,(1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl, et al., (1992)Science 225:1570-1573); WIP1 (Rohmeier, et al., (1993) Plant Mol. Biol.22:783-792; Eckelkamp, et al., (1993) FEBS Letters 323:73-76); MPI gene(Corderok, et al., (1994) Plant J. 6(2):141-150) and the like, hereinincorporated by reference.

Additionally, pathogen-inducible promoters may be employed in themethods and nucleotide constructs of the embodiments. Suchpathogen-inducible promoters include those from pathogenesis-relatedproteins (PR proteins), which are induced following infection by apathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,chitinase, etc. See, for example, Redolfi, et al., (1983) Neth. J. PlantPathol. 89:245-254; Uknes, et al., (1992) Plant Cell 4: 645-656 and VanLoon, (1985) Plant Mol. Virol. 4:111-116. See also, WO 1999/43819,herein incorporated by reference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau, et al., (1987) PlantMol. Biol. 9:335-342; Matton, et al., (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch, et al., (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. 2:93-98 andYang, (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen,et al., (1996) Plant J. 10:955-966; Zhang, et al., (1994) Proc. Natl.Acad. Sci. USA 91:2507-2511; Warner, et al., (1993) Plant J. 3:191-201;Siebertz, et al., (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible) and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero, et al., (1992) Physiol. Mol. Plant Path. 41:189-200).

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression or a chemical-repressible promoter, where application ofthe chemical represses gene expression. Chemical-inducible promoters areknown in the art and include, but are not limited to, the maize In2-2promoter, which is activated by benzenesulfonamide herbicide safeners,the maize GST promoter, which is activated by hydrophobic electrophiliccompounds that are used as pre-emergent herbicides, and the tobaccoPR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena, et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis, et al., (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237 and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced an IPD090polypeptide expression within a particular plant tissue.Tissue-preferred promoters include those discussed in Yamamoto, et al.,(1997) Plant J. 12(2)255-265; Kawamata, et al., (1997) Plant CellPhysiol. 38(7):792-803; Hansen, et al., (1997) Mol. Gen Genet.254(3):337-343; Russell, et al., (1997) Transgenic Res. 6(2):157-168;Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341; Van Camp, etal., (1996) Plant Physiol. 112(2):525-535; Canevascini, et al., (1996)Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994) Plant CellPhysiol. 35(5):773-778; Lam, (1994) Results Probl. Cell Differ.20:181-196; Orozco, et al., (1993) Plant Mol Biol. 23(6):1129-1138;Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 andGuevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Additional tissuespecific promoters are known in the art including the promoters of USPatent Numbers U.S. Pat. Nos. 8,816,152 and 9,150,624. Such promoterscan be modified, if necessary, for weak expression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al., (1994)Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant Cell Physiol.35(5):773-778; Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al.,(1993) Plant Mol. Biol. 23(6):1129-1138 and Matsuoka, et al., (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

US Patent Application-preferred or root-specific promoters are known andcan be selected from the many available from the literature or isolatedde novo from various compatible species. See, for example, Hire, et al.,(1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutaminesynthetase gene); Keller and Baumgartner, (1991) Plant Cell3(10):1051-1061 (root-specific control element in the GRP 1.8 gene ofFrench bean); Sanger, et al., (1990) Plant Mol. Biol. 14(3):433-443(root-specific promoter of the mannopine synthase (MAS) gene ofAgrobacterium tumefaciens) and Miao, et al., (1991) Plant Cell3(1):11-22 (full-length cDNA clone encoding cytosolic glutaminesynthetase (GS), which is expressed in roots and root nodules ofsoybean). See also, Bogusz, et al., (1990) Plant Cell 2(7):633-641,where two root-specific promoters isolated from hemoglobin genes fromthe nitrogen-fixing nonlegume Parasponia andersonii and the relatednon-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a 3-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi, (1991) describe their analysisof the promoters of the highly expressed roIC and roID root-inducinggenes of Agrobacterium rhizogenes (see, Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri, et al., (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see, EMBO J. 8(2):343-350). The TR1′ gene fused tonptII (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster, et al., (1995) Plant Mol. Biol. 29(4):759-772) androlB promoter (Capana, et al., (1994) Plant Mol. Biol. 25(4):681-691.See also, U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252;5,401,836; 5,110,732 and 5,023,179. Arabidopsis thaliana root-preferredregulatory sequences are disclosed in US20130117883. US PatentApplication Publication Number US20160097054 discloses the sorghumroot-preferred promoter PLTP. US Patent Application Publication NumberUS20160145634 discloses the sorghum root-preferred promoter TIP2-3. U.S.Pat. No. 8,916,377 discloses the sorghum root-preferred promoter RCc3.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See, Thompson, et al., (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); and milps(myo-inositol-1-phosphate synthase) (see, U.S. Pat. No. 6,225,529,herein incorporated by reference). Gamma-zein and Glb-1 areendosperm-specific promoters. For dicots, seed-specific promotersinclude, but are not limited to, Kunitz trypsin inhibitor 3 (KTi3)(Jofuku and Goldberg, (1989) Plant Cell 1:1079-1093), bean β-phaseolin,napin, β-conglycinin, glycinin 1, soybean lectin, cruciferin, and thelike. For monocots, seed-specific promoters include, but are not limitedto, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken1, shrunken 2, globulin 1, etc. See also, WO 2000/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed; hereinincorporated by reference. In dicots, seed specific promoters includebut are not limited to seed coat promoter from Arabidopsis, pBAN; andthe early seed promoters from Arabidopsis, p26, p63, and p63tr (U.S.Pat. Nos. 7,294,760 and 7,847,153). A promoter that has “preferred”expression in a particular tissue is expressed in that tissue to agreater degree than in at least one other plant tissue. Sometissue-preferred promoters show expression almost exclusively in theparticular tissue.

Where low level expression is desired, weak promoters will be used.Generally, the term “weak promoter” as used herein refers to a promoterthat drives expression of a coding sequence at a low level. By low levelexpression at levels of between about 1/1000 transcripts to about1/100,000 transcripts to about 1/500,000 transcripts is intended.Alternatively, it is recognized that the term “weak promoters” alsoencompasses promoters that drive expression in only a few cells and notin others to give a total low level of expression. Where a promoterdrives expression at unacceptably high levels, portions of the promotersequence can be deleted or modified to decrease expression levels.

Such weak constitutive promoters include, for example the core promoterof the Rsyn7 promoter (WO 1999/43838 and U.S. Pat. No. 6,072,050), thecore 35S CaMV promoter, and the like. Other constitutive promotersinclude, for example, those disclosed in U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;5,608,142, 6,177,611, and 8,697,857, herein incorporated by reference.

Chimeric or hybrid promoters are also known in art including thosedisclosed in US Patent Numbers U.S. Pat. Nos. 8,846,892, 8,822,666, and9,181,560.

The above list of promoters is not meant to be limiting. Any appropriatepromoter can be used in the embodiments.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glufosinate ammonium, bromoxynil, imidazolinones and2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitableselectable marker genes include, but are not limited to, genes encodingresistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J.2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature303:209-213 and Meijer, et al., (1991) Plant Mol. Biol. 16:807-820);streptomycin (Jones, et al., (1987) Mol. Gen. Genet. 210:86-91);spectinomycin (Bretagne-Sagnard, et al., (1996) Transgenic Res.5:131-137); bleomycin (Hille, et al., (1990) Plant Mol. Biol.7:171-176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol.15:127-136); bromoxynil (Stalker, et al., (1988) Science 242:419-423);glyphosate (Shaw, et al., (1986) Science 233:478-481 and U.S. patentapplication Ser. Nos. 10/004,357 and 10/427,692); phosphinothricin(DeBlock, et al., (1987) EMBO J. 6:2513-2518). See generally, Yarranton,(1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992)Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao, et al., (1992) Cell71:63-72; Reznikoff, (1992) Mol. Microbiol. 6:2419-2422; Barkley, etal., (1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell48:555-566; Brown, et al., (1987) Cell 49:603-612; Figge, et al., (1988)Cell 52:713-722; Deuschle, et al., (1989) Proc. Natl. Acad. Sci. USA86:5400-5404; Fuerst, et al., (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle, et al., (1990) Science 248:480-483; Gossen,(1993) Ph.D. Thesis, University of Heidelberg; Reines, et al., (1993)Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol.Cell. Biol. 10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad.Sci. USA 89:3952-3956; Baim, et al., (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10:143-162;Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35:1591-1595;Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993)Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob.Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook ofExperimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin) and Gill,et al., (1988) Nature 334:721-724. Such disclosures are hereinincorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the embodiments.

Plant Transformation

The methods of the embodiments involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is as used herein meanspresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the embodiments do not depend on a particularmethod for introducing a polynucleotide or polypeptide into a plant,only that the polynucleotide or polypeptides gains access to theinterior of at least one cell of the plant. Methods for introducingpolynucleotide or polypeptides into plants are known in the artincluding, but not limited to, stable transformation methods, transienttransformation methods, and virus-mediated methods.

“Stable transformation” is as used herein means that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” as used herein means that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant. “Plant” as usedherein refers to whole plants, plant organs (e.g., leaves, stems, roots,etc.), seeds, plant cells, propagules, embryos and progeny of the same.Plant cells can be differentiated or undifferentiated (e.g. callus,suspension culture cells, protoplasts, leaf cells, root cells, phloemcells and pollen).

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway, et al., (1986) Biotechniques 4:320-334), electroporation(Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606),Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J.3:2717-2722) and ballistic particle acceleration (see, for example, U.S.Pat. Nos. 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes, et al.,(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,ed. Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe, et al.,(1988) Biotechnology 6:923-926) and Led transformation (WO 00/28058).For potato transformation see, Tu, et al., (1998) Plant MolecularBiology 37:829-838 and Chong, et al., (2000) Transgenic Research9:71-78. Additional transformation procedures can be found inWeissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al.,(1987) Particulate Science and Technology 5:27-37 (onion); Christou, etal., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor.Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563(maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, etal., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984)Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals);Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman, et al., (Longman, New York), pp. 197-209(pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 andKaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413(rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, the sequences of the embodiments can beprovided to a plant using a variety of transient transformation methods.Such transient transformation methods include, but are not limited to,the introduction of the IPD090 polynucleotide or variants and fragmentsthereof directly into the plant or the introduction of the IPD090polypeptide transcript into the plant. Such methods include, forexample, microinjection or particle bombardment. See, for example,Crossway, et al., (1986) Mol Gen. Genet. 202:179-185; Nomura, et al.,(1986) Plant Sci. 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad.Sci. 91:2176-2180 and Hush, et al., (1994) The Journal of Cell Science107:775-784, all of which are herein incorporated by reference.Alternatively, the IPD090 polynucleotide can be transiently transformedinto the plant using techniques known in the art. Such techniquesinclude viral vector system and the precipitation of the polynucleotidein a manner that precludes subsequent release of the DNA. Thus,transcription from the particle-bound DNA can occur, but the frequencywith which it is released to become integrated into the genome isgreatly reduced. Such methods include the use of particles coated withpolyethylimine (PEI; Sigma #P3143).

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO 1999/25855and WO 1999/25853, all of which are herein incorporated by reference.Briefly, the polynucleotide of the embodiments can be contained intransfer cassette flanked by two non-identical recombination sites. Thetransfer cassette is introduced into a plant have stably incorporatedinto its genome a target site which is flanked by two non-identicalrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome.

Plant transformation vectors may be comprised of one or more DNA vectorsneeded for achieving plant transformation. For example, it is a commonpractice in the art to utilize plant transformation vectors that arecomprised of more than one contiguous DNA segment. These vectors areoften referred to in the art as “binary vectors”. Binary vectors as wellas vectors with helper plasmids are most often used forAgrobacterium-mediated transformation, where the size and complexity ofDNA segments needed to achieve efficient transformation is quite large,and it is advantageous to separate functions onto separate DNAmolecules. Binary vectors typically contain a plasmid vector thatcontains the cis-acting sequences required for T-DNA transfer (such asleft border and right border), a selectable marker that is engineered tobe capable of expression in a plant cell, and a “gene of interest” (agene engineered to be capable of expression in a plant cell for whichgeneration of transgenic plants is desired). Also present on thisplasmid vector are sequences required for bacterial replication. Thecis-acting sequences are arranged in a fashion to allow efficienttransfer into plant cells and expression therein. For example, theselectable marker gene and the pesticidal gene are located between theleft and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as is understood in the art (Hellens and Mullineaux, (2000)Trends in Plant Science 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g., immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Following integration of heterologous foreign DNA into plant cells, onethen applies a maximum threshold level of appropriate selection in themedium to kill the untransformed cells and separate and proliferate theputatively transformed cells that survive from this selection treatmentby transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one identifies and proliferatesthe cells that are transformed with the plasmid vector. Molecular andbiochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest into the genome of thetransgenic plant.

Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g., Hiei, et al., (1994) ThePlant Journal 6:271-282; Ishida, et al., (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park, (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar, (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick, et al.,(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive or inducible expression ofthe desired phenotypic characteristic identified. Two or moregenerations may be grown to ensure that expression of the desiredphenotypic characteristic is stably maintained and inherited and thenseeds harvested to ensure that expression of the desired phenotypiccharacteristic has been achieved.

The nucleotide sequences of the embodiments may be provided to the plantby contacting the plant with a virus or viral nucleic acids. Generally,such methods involve incorporating the nucleotide construct of interestwithin a viral DNA or RNA molecule. It is recognized that therecombinant proteins of the embodiments may be initially synthesized aspart of a viral polyprotein, which later may be processed by proteolysisin vivo or in vitro to produce the desired IPD090 polypeptide. It isalso recognized that such a viral polyprotein, comprising at least aportion of the amino acid sequence of an IPD090 of the embodiments, mayhave the desired pesticidal activity. Such viral polyproteins and thenucleotide sequences that encode for them are encompassed by theembodiments. Methods for providing plants with nucleotide constructs andproducing the encoded proteins in the plants, which involve viral DNA orRNA molecules, are known in the art. See, for example, U.S. Pat. Nos.5,889,191; 5,889,190; 5,866,785; 5,589,367 and 5,316,931; hereinincorporated by reference.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab, et al., (1990) Proc. Natl. Acad. Sci. USA87:8526-8530; Svab and Maliga, (1993) Proc. Natl. Acad. Sci. USA90:913-917; Svab and Maliga, (1993) EMBO J. 12:601-606. The methodrelies on particle gun delivery of DNA containing a selectable markerand targeting of the DNA to the plastid genome through homologousrecombination. Additionally, plastid transformation can be accomplishedby transactivation of a silent plastid-borne transgene bytissue-preferred expression of a nuclear-encoded and plastid-directedRNA polymerase. Such a system has been reported in McBride, et al.,(1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

The embodiments further relate to plant-propagating material of atransformed plant of the embodiments including, but not limited to,seeds, tubers, corms, bulbs, leaves and cuttings of roots and shoots.

The embodiments may be used for transformation of any plant species,including, but not limited to, monocots and dicots. Examples of plantsof interest include, but are not limited to, corn (Zea mays), Brassicasp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the embodiments include, for example, pines suchas loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosapine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Montereypine (Pinus radiata); Douglas-fir (Pseudotsuga menziesi); Westernhemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Plants of the embodiments include crop plants (for example, corn,alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut,sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

Turf grasses include, but are not limited to: annual bluegrass (Poaannua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poacompressa); Chewing's fescue (Festuca rubra); colonial bentgrass(Agrostis tenuis); creeping bentgrass (Agrostis palustris); crestedwheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyroncristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poapratensis); orchardgrass (Dactylis glomerata); perennial ryegrass(Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba);rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smoothbromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy(Phleum pratense); velvet bentgrass (Agrostis canina); weepingalkaligrass (Puccinellia distans); western wheatgrass (Agropyronsmithii); Bermuda grass (Cynodon spp.); St. Augustine grass(Stenotaphrum secundatum); zoysia grass (Zoysia spp.); Bahia grass(Paspalum notatum); carpet grass (Axonopus affinis); centipede grass(Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum);seashore paspalum (Paspalum vaginatum); blue gramma (Boutelouagracilis); buffalo grass (Buchloe dactyloids); sideoats gramma(Bouteloua curtipendula).

Plants of interest include grain plants that provide seeds of interest,oil-seed plants, and leguminous plants. Seeds of interest include grainseeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc.Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica,maize, alfalfa, palm, coconut, flax, castor, olive, etc. Leguminousplants include beans and peas. Beans include guar, locust bean,fenugreek, soybean, garden beans, cowpea, mung bean, lima bean, favabean, lentils, chickpea, etc.

Evaluation of Plant Transformation

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of heterologous gene in the plant genomeis confirmed by various methods such as analysis of nucleic acids,proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell, (2001) MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, (2001) supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” is then probedwith, for example, radiolabeled 32P target DNA fragment to confirm theintegration of introduced gene into the plant genome according tostandard techniques (Sambrook and Russell, (2001) supra).

In Northern blot analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, and blottedonto a nylon filter according to standard procedures that are routinelyused in the art (Sambrook and Russell, (2001) supra). Expression of RNAencoded by the pesticidal gene is then tested by hybridizing the filterto a radioactive probe derived from a pesticidal gene, by methods knownin the art (Sambrook and Russell, (2001) supra).

Western blot, biochemical assays and the like may be carried out on thetransgenic plants to confirm the presence of protein encoded by thepesticidal gene by standard procedures (Sambrook and Russell, 2001,supra) using antibodies that bind to one or more epitopes present on theIPD090 polypeptide.

Methods to Introduce Genome Editing Technologies into Plants

In some embodiments, the disclosed IPD090 polynucleotide compositionscan be introduced into the genome of a plant using genome editingtechnologies, or previously introduced IPD090 polynucleotides in thegenome of a plant may be edited using genome editing technologies. Forexample, the disclosed polynucleotides can be introduced into a desiredlocation in the genome of a plant through the use of double-strandedbreak technologies such as TALENs, meganucleases, zinc finger nucleases,CRISPR-Cas, and the like. For example, the disclosed polynucleotides canbe introduced into a desired location in a genome using a CRISPR-Cassystem, for the purpose of site-specific insertion. The desired locationin a plant genome can be any desired target site for insertion, such asa genomic region amenable for breeding or may be a target site locatedin a genomic window with an existing trait of interest. Existing traitsof interest could be either an endogenous trait or a previouslyintroduced trait.

In some embodiments, where the disclosed IPD090 polynucleotide haspreviously been introduced into a genome, genome editing technologiesmay be used to alter or modify the introduced polynucleotide sequence.Site specific modifications that can be introduced into the disclosedIPD090 polynucleotide compositions include those produced using anymethod for introducing site specific modification, including, but notlimited to, through the use of gene repair oligonucleotides (e.g. USPublication 2013/0019349), or through the use of double-stranded breaktechnologies such as TALENs, meganucleases, zinc finger nucleases,CRISPR-Cas, and the like. Such technologies can be used to modify thepreviously introduced polynucleotide through the insertion, deletion orsubstitution of nucleotides within the introduced polynucleotide.Alternatively, double-stranded break technologies can be used to addadditional nucleotide sequences to the introduced polynucleotide.Additional sequences that may be added include, additional expressionelements, such as enhancer and promoter sequences. In anotherembodiment, genome editing technologies may be used to positionadditional insecticidally-active proteins in close proximity to thedisclosed IPD090 polynucleotide compositions disclosed herein within thegenome of a plant, in order to generate molecular stacks ofinsecticidally-active proteins.

An “altered target site,” “altered target sequence.” “modified targetsite,” and “modified target sequence” are used interchangeably hereinand refer to a target sequence as disclosed herein that comprises atleast one alteration when compared to non-altered target sequence. Such“alterations” include, for example: (i) replacement of at least onenucleotide, (ii) a deletion of at least one nucleotide, (iii) aninsertion of at least one nucleotide, or (iv) any combination of(i)-(iii).

Stacking of Traits in Transgenic Plant

Transgenic plants may comprise a stack of one or more insecticidalpolynucleotides disclosed herein with one or more additionalpolynucleotides resulting in the production or suppression of multiplepolypeptide sequences. Transgenic plants comprising stacks ofpolynucleotide sequences can be obtained by either or both oftraditional breeding methods or through genetic engineering methods.These methods include, but are not limited to, breeding individual lineseach comprising a polynucleotide of interest, transforming a transgenicplant comprising a gene disclosed herein with a subsequent gene andco-transformation of genes into a single plant cell. As used herein, theterm “stacked” includes having the multiple traits present in the sameplant (i.e., both traits are incorporated into the nuclear genome, onetrait is incorporated into the nuclear genome and one trait isincorporated into the genome of a plastid or both traits areincorporated into the genome of a plastid). In one non-limiting example,“stacked traits” comprise a molecular stack where the sequences arephysically adjacent to each other. A trait, as used herein, refers tothe phenotype derived from a particular sequence or groups of sequences.Co-transformation of genes can be carried out using singletransformation vectors comprising multiple genes or genes carriedseparately on multiple vectors. If the sequences are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. The traits can beintroduced simultaneously in a co-transformation protocol with thepolynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO1999/25855 and WO 1999/25853, all of which are herein incorporated byreference.

In some embodiments the polynucleotides encoding the IPD090 polypeptidedisclosed herein, alone or stacked with one or more additional insectresistance traits can be stacked with one or more additional inputtraits (e.g., herbicide resistance, fungal resistance, virus resistance,stress tolerance, disease resistance, male sterility, stalk strength,and the like) or output traits (e.g., increased yield, modifiedstarches, improved oil profile, balanced amino acids, high lysine ormethionine, increased digestibility, improved fiber quality, droughtresistance, and the like). Thus, the polynucleotide embodiments can beused to provide a complete agronomic package of improved crop qualitywith the ability to flexibly and cost effectively control any number ofagronomic pests.

Transgenes useful for stacking include but are not limited to:

1. Transgenes that Confer Resistance to Insects or Disease and thatEncode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones, et al., (1994) Science266:789 (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin, et al., (1993) Science 262:1432 (tomato Pto gene forresistance to Pseudomonas syringae pv. tomato encodes a protein kinase);Mindrinos, et al., (1994) Cell 78:1089 (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae), McDowell and Woffenden, (2003)Trends Biotechnol. 21(4):178-83 and Toyoda, et al., (2002) TransgenicRes. 11(6):567-82. A plant resistant to a disease is one that is moreresistant to a pathogen as compared to the wild type plant.

(B) Genes encoding a Bacillus thuringiensis protein, a derivativethereof or a synthetic polypeptide modeled thereon. See, for example,Geiser, et al., (1986) Gene 48:109, who disclose the cloning andnucleotide sequence of a Bt delta-endotoxin gene. Moreover, DNAmolecules encoding delta-endotoxin genes can be purchased from AmericanType Culture Collection (Rockville, Md.), for example, under ATCC®Accession Numbers 40098, 67136, 31995 and 31998. Other non-limitingexamples of Bacillus thuringiensis transgenes being geneticallyengineered are given in the following patents and patent applicationsand hereby are incorporated by reference for this purpose: U.S. Pat.Nos. 5,188,960; 5,689,052; 5,880,275; 5,986,177; 6,023,013, 6,060,594,6,063,597, 6,077,824, 6,620,988, 6,642,030, 6,713,259, 6,893,826,7,105,332; 7,179,965, 7,208,474; 7,227,056, 7,288,643, 7,323,556,7,329,736, 7,449,552, 7,468,278, 7,510,878, 7,521,235, 7,544,862,7,605,304, 7,696,412, 7,629,504, 7,705,216, 7,772,465, 7,790,846,7,858,849, 9,546,378; US Patent Publication US20160376607 and WO1991/14778; WO 1999/31248; WO 2001/12731; WO 1999/24581 and WO1997/40162.

Genes encoding pesticidal proteins may also be stacked including but arenot limited to: insecticidal proteins from Pseudomonas sp. such asPSEEN3174 (Monalysin, (2011) PLoS Pathogens, 7:1-13), from Pseudomonasprotegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr,(2008) Environmental Microbiology 10:2368-2386: GenBank Accession No.EU400157); from Pseudomonas taiwanensis (Liu, et al., (2010) J. Agric.Food Chem. 58:12343-12349) and from Pseudomonas pseudoalcaligenes(Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li, et al.,(2007) Plant Cell Tiss. Organ Cult. 89:159-168); insecticidal proteinsfrom Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010)The Open Toxinology Journal 3:101-118 and Morgan, et al., (2001) Appliedand Envir. Micro. 67:2062-2069), U.S. Pat. Nos. 6,048,838, and6,379,946; a PIP-1 polypeptide of US Patent Publication US20140007292;an AflP-1A and/or AflP-1B polypeptide of US Patent PublicationUS20140033361; a PHI-4 polypeptide of US Patent PublicationUS20140274885 and US20160040184; a PIP-47 polypeptide of PCT PublicationNumber WO2015/023846, a PIP-72 polypeptide of US Publication NumberUS20160366891; a PtlP-50 polypeptide and a PtlP-65 polypeptide of PCTPublication Number WO2015/120270; a PtlP-83 polypeptide of PCTPublication Number WO2015/120276; a PtlP-96 polypeptide of PCT SerialNumber PCT/US15/55502; an IPD079 polypeptide of PCT Publication NumberWO2017/023486; an IPD082 polypeptide of Serial Number PCT/US16/65531; anIPD093 polypeptide of U.S. Ser. No. 62/434,020; an IPD080 polypeptide ofUS Serial Number U.S. 62/411,318; and δ-endotoxins including, but notlimited to, the Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9,Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19,Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry28, Cry29,Cry30, Cry31, Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, Cry39,Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry46, Cry47, Cry49, Cry50,Cry51, Cry52, Cry53, Cry54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60,Cry61, Cry62, Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70,Cry71, and Cry72 classes of δ-endotoxin genes and the B. thuringiensiscytolytic Cyt1 and Cyt2 genes. Members of these classes of B.thuringiensis insecticidal proteins well known to one skilled in the art(see, Crickmore, et al., “Bacillus thuringiensis toxin nomenclature”(2011), at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can beaccessed on the world-wide web using the “www” prefix).

Examples of δ-endotoxins also include but are not limited to Cry1Aproteins of U.S. Pat. Nos. 5,880,275, 7,858,849 and 8,878,007; a Cry1Acmutant of U.S. Pat. No. 9,512,187; a DIG-3 or DIG-11 toxin (N-terminaldeletion of α-helix 1 and/or α-helix 2 variants of Cry proteins such asCry1A) of U.S. Pat. Nos. 8,304,604 and 8,304,605, a DIG-10 of U.S. Pat.No. 8,697,857; Cry1B of U.S. patent application Ser. No. 10/525,318, USPatent Application Publication Number US20160194364, and U.S. Pat. Nos.9,404,121 and 8,772,577; Cry1B variants of PCT Publication NumberWO2016/61197 and Serial Number PCT/US17/27160; Cry1C of U.S. Pat. No.6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960, 6,218,188; Cry1A/Fchimeras of U.S. Pat. Nos. 7,070,982; 6,962,705 and 6,713,063); a Cry2protein such as Cry2Ab protein of U.S. Pat. No. 7,064,249); a Cry3Aprotein including but not limited to an engineered hybrid insecticidalprotein (eHIP) created by fusing unique combinations of variable regionsand conserved blocks of at least two different Cry proteins such asCry3A with Cry1Aa or Cry1Ab (U.S. Pat. Nos. 8,309,516 and 9,522,937); aCry4 protein; a Cry5 protein; a Cry6 protein; Cry8 proteins of U.S. Pat.Nos. 7,329,736, 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,339,0927,378,499 and 7,462,760; a Cry9 protein such as such as members of theCry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families including the Cry9proteins of U.S. Pat. Nos. 9,000,261 and 8,802,933, and U.S. Ser. No.62/287,281; a Cry15 protein of Naimov, et al., (2008) Applied andEnvironmental Microbiology 74:7145-7151; a Cry22, a Cry34Ab1 protein ofU.S. Pat. Nos. 6,127,180, 6,624,145 and 6,340,593; a truncated Cry34protein of U.S. Pat. No. 8,816,157; a CryET33 and CryET34 protein ofU.S. Pat. Nos. 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107 and7,504,229; a CryET33 and CryET34 homologs of US Patent PublicationNumber 2006/0191034, 2012/0278954, and PCT Publication Number WO2012/139004; a Cry35Ab1 protein of U.S. Pat. Nos. 6,083,499, 6,548,291and 6,340,593; a Cry46 protein of U.S. Pat. No. 9,403,881, a Cry51protein, a Cry binary toxin; a TIC901 or related toxin; TIC807 of US2008/0295207; TIC853 of US Patent U.S. Pat. No. 8,513,493; ET29, ET37,TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US 2006/033867; engineeredHemipteran toxic proteins of US Patent Application Publication NumberUS20160150795; TIC1498, TIC1415, TIC1497, TIC1886, TIC1925, TIC1414,TIC1885, TIC1922, TIC1422, TIC 1974, TIC2032, TIC2120, TIC1362 of USPatent U.S. Pat. No. 9,238,678; a TIC2463-type protein of US PatentApplication Publication Number US20150274786; TIC3668-type protein of USPatent Application Publication Number US20160319302; AXMI-027, AXMI-036,and AXMI-038 of U.S. Pat. No. 8,236,757; AXMI-031, AXMI-039, AXMI-040,AXMI-049 of U.S. Pat. No. 7,923,602; AXMI-018, AXMI-020, and AXMI-021 ofWO 2006/083891; AXMI-010 of WO 2005/038032; AXMI-003 of WO 2005/021585;AXMI-008 of US 2004/0250311; AXMI-006 of US 2004/0216186; AXMI-007 of US2004/0210965; AXMI-009 of US 2004/0210964; AXMI-014 of US 2004/0197917;AXMI-004 of US 2004/0197916; AXMI-028 and AXMI-029 of WO 2006/119457;AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 ofUS20110023184; AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019,AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023,AXMI-041, AXMI-063, and AXMI-064 of US 2011/0263488; AXMI-R1 and relatedproteins of US 2010/0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z andAXMI225z of WO 2011/103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227,AXMI228, AXMI229, AXMI230, and AXMI231 of U.S. Pat. No. 9,156,895;AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No.8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI-035, and AXMI-045 of US2010/0298211; AXMI-066 and AXMI-076 of US2009/0144852; AXMI128, AXMI130,AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146,AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157,AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170,AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178,AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188,AXMI189 of U.S. Pat. No. 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082,AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101,AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111,AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121,AXMI122, AXMI123, AXMI124, AXMI125, AXMI126, AXMI127, AXMI129, AXMI164,AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of US U.S. Pat. No.8,461,421; AXMI192 of US Patent U.S. Pat. No. 8,461,415; AXMI234 andAXMI235 of US Patent Application Publication Number US20150218583;AXMI281 of US Patent Application Publication Number US20160177332;AXMI422 of U.S. Pat. No. 8,252,872; and Cry proteins such as Cry1A andCry3A having modified proteolytic sites of U.S. Pat. No. 8,319,019; amodified Cry3 of U.S. Pat. No. 9,109,231; and a Cry1Ac, Cry2Aa and Cry1Ca toxin protein from Bacillus thuringiensis strain VBTS 2528 of USPatent Application Publication Number 2011/0064710. The Cry proteinsMP032, MP049, MP051, MP066, MP068, MP070, MP091S, MP109S, MP114, MP121,MP134S, MP183S, MP185S, MP186S, MP195S, MP197S, MP208S, MP209S, MP212S,MP214S, MP217S, MP222S, MP234S, MP235S, MP237S, MP242S, MP243, MP248,MP249S, MP251M, MP252S, MP253, MP259S, MP287S, MP288S, MP295S, MP296S,MP297S, MP300S, MP304S, MP306S, MP310S, MP312S, MP314S, MP319S, MP325S,MP326S, MP327S, MP328S, MP334S, MP337S, MP342S, MP349S, MP356S, MP359S,MP360S, MP437S, MP451S, MP452S, MP466S, MP468S, MP476S, MP482S, MP522S,MP529S, MP548S, MP552S, MP562S, MP564S, MP566S, MP567S, MP569S, MP573S,MP574S, MP575S, MP581S, MP590, MP594S, MP596S, MP597, MP599S, MP600S,MP601S, MP602S, MP604S, MP626S, MP629S, MP630S, MP631S, MP632S, MP633S,MP634S, MP635S, MP639S, MP640S, MP644S, MP649S, MP651S, MP652S, MP653S,MP661S, MP666S, MP672S, MP696S, MP704S, MP724S, MP729S, MP739S, MP755S,MP773S, MP799S, MP800S, MP801S, MP802S, MP803S, MP805S, MP809S, MP815S,MP828S, MP831S, MP844S, MP852, MP865S, MP879S, MP887S, MP891S, MP896S,MP898S, MP935S, MP968, MP989, MP993, MP997, MP1049, MP1066, MP1067,MP1080, MP1081, MP1200, MP1206, MP1233, and MP1311 of U.S. Ser. No.62/429,426. Other Cry proteins are well known to one skilled in the art(see, Crickmore, et al., “Bacillus thuringiensis toxin nomenclature”(2011), at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can beaccessed on the world-wide web using the “www” prefix). The insecticidalactivity of Cry proteins is well known to one skilled in the art (forreview, see, van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16). Theuse of Cry proteins as transgenic plant traits is well known to oneskilled in the art and Cry-transgenic plants including but not limitedto Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2,Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A (US Patent U.S. Pat. No. 7,276,583),Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A (US Patent U.S. Pat. No.7,276,583), Cry9c and CBI-Bt have received regulatory approval (see,Sanahuja, (2011) Plant Biotech Journal 9:283-300 and the CERA (2010) GMCrop Database Center for Environmental Risk Assessment (CERA), ILSIResearch Foundation, Washington D.C. atcera-gmc.org/index.php?action=gm_crop_database which can be accessed onthe world-wide web using the “www” prefix). More than one pesticidalproteins well known to one skilled in the art can also be expressed inplants such as Vip3Ab & Cry1Fa (US2012/0317682), Cry1BE & Cry1F(US2012/0311746), Cry1CA & Cry1AB (US2012/0311745), Cry1F & CryCa(US2012/0317681), Cry1DA & Cry1BE (US2012/0331590), Cry1DA & Cry1Fa(US2012/0331589), Cry1AB & Cry1BE (US2012/0324606), and Cry1Fa & Cry2Aa,Cry1I or Cry1E (US2012/0324605). Pesticidal proteins also includeinsecticidal lipases including lipid acyl hydrolases of U.S. Pat. No.7,491,869, and cholesterol oxidases such as from Streptomyces (Purcellet al. (1993) Biochem Biophys Res Commun 15:1406-1413). Pesticidalproteins also include VIP (vegetative insecticidal proteins) toxins ofU.S. Pat. Nos. 5,877,012, 6,107,279, 6,137,033, 7,244,820, 7,615,686,and 8,237,020, and the like. Other VIP proteins are well known to oneskilled in the art (see,lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html which can beaccessed on the world-wide web using the “www” prefix). Pesticidalproteins also include toxin complex (TC) proteins, obtainable fromorganisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, U.S.Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have “stand alone”insecticidal activity and other TC proteins enhance the activity of thestand-alone toxins produced by the same given organism. The toxicity ofa “stand-alone” TC protein (from Photorhabdus, Xenorhabdus orPaenibacillus, for example) can be enhanced by one or more TC protein“potentiators” derived from a source organism of a different genus.There are three main types of TC proteins. As referred to herein, ClassA proteins (“Protein A”) are stand-alone toxins. Class B proteins(“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity ofClass A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 andXptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi.Examples of Class C proteins are TccC, XptC1Xb and XptB1Wi. Pesticidalproteins also include spider, snake and scorpion venom proteins.Examples of spider venom peptides include but are not limited tolycotoxin-1 peptides and mutants thereof (U.S. Pat. No. 8,334,366).

(C) A polynucleotide encoding an insect-specific hormone or pheromonesuch as an ecdysteroid and juvenile hormone, a variant thereof, amimetic based thereon or an antagonist or agonist thereof. See, forexample, the disclosure by Hammock, et al., (1990) Nature 344:458, ofbaculovirus expression of cloned juvenile hormone esterase, aninactivator of juvenile hormone.

(D) A polynucleotide encoding an insect-specific peptide which, uponexpression, disrupts the physiology of the affected pest. For example,see the disclosures of, Regan, (1994) J. Biol. Chem. 269:9 (expressioncloning yields DNA coding for insect diuretic hormone receptor); Pratt,et al., (1989) Biochem. Biophys. Res. Comm. 163:1243 (an allostatin isidentified in Diploptera puntata); Chattopadhyay, et al., (2004)Critical Reviews in Microbiology 30(1):33-54; Zjawiony, (2004) J NatProd 67(2):300-310; Carlini and Grossi-de-Sa, (2002) Toxicon40(11):1515-1539; Ussuf, et al., (2001) Curr Sci. 80(7):847-853 andVasconcelos and Oliveira, (2004) Toxicon 44(4):385-403. See also, U.S.Pat. No. 5,266,317 to Tomalski, et al., who disclose genes encodinginsect-specific toxins.

(E) A polynucleotide encoding an enzyme responsible for ahyperaccumulation of a monoterpene, a sesquiterpene, a steroid,hydroxamic acid, a phenylpropanoid derivative or another non-proteinmolecule with insecticidal activity.

(F) A polynucleotide encoding an enzyme involved in the modification,including the post-translational modification, of a biologically activemolecule; for example, a glycolytic enzyme, a proteolytic enzyme, alipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, ahydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, anelastase, a chitinase and a glucanase, whether natural or synthetic.See, PCT Application WO 1993/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene. DNA molecules whichcontain chitinase-encoding sequences can be obtained, for example, fromthe ATCC® under Accession Numbers 39637 and 67152. See also, Kramer, etal., (1993) Insect Biochem. Molec. Biol. 23:691, who teach thenucleotide sequence of a cDNA encoding tobacco hookworm chitinase andKawalleck, et al., (1993) Plant Molec. Biol. 21:673, who provide thenucleotide sequence of the parsley ubi4-2 polyubiquitin gene, and U.S.Pat. Nos. 6,563,020; 7,145,060 and 7,087,810.

(G) A polynucleotide encoding a molecule that stimulates signaltransduction. For example, see the disclosure by Botella, et al., (1994)Plant Molec. Biol. 24:757, of nucleotide sequences for mung beancalmodulin cDNA clones, and Griess, et al., (1994) Plant Physiol.104:1467, who provide the nucleotide sequence of a maize calmodulin cDNAclone.

(H) A polynucleotide encoding a hydrophobic moment peptide. See, PCTApplication WO 1995/16776 and U.S. Pat. No. 5,580,852 disclosure ofpeptide derivatives of Tachyplesin which inhibit fungal plant pathogens)and PCT Application WO 1995/18855 and U.S. Pat. No. 5,607,914 (teachessynthetic antimicrobial peptides that confer disease resistance).

(I) A polynucleotide encoding a membrane permease, a channel former or achannel blocker. For example, see the disclosure by Jaynes, et al.,(1993) Plant Sci. 89:43, of heterologous expression of a cecropin-betalytic peptide analog to render transgenic tobacco plants resistant toPseudomonas solanacearum.

(J) A gene encoding a viral-invasive protein or a complex toxin derivedtherefrom. For example, the accumulation of viral coat proteins intransformed plant cells imparts resistance to viral infection and/ordisease development effected by the virus from which the coat proteingene is derived, as well as by related viruses. See, Beachy, et al.,(1990) Ann. Rev. Phytopathol. 28:451. Coat protein-mediated resistancehas been conferred upon transformed plants against alfalfa mosaic virus,cucumber mosaic virus, tobacco streak virus, potato virus X, potatovirus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaicvirus. Id.

(K) A gene encoding an insect-specific antibody or an immunotoxinderived therefrom. Thus, an antibody targeted to a critical metabolicfunction in the insect gut would inactivate an affected enzyme, killingthe insect. Cf. Taylor, et al., Abstract #497, SEVENTH INT'L SYMPOSIUMON MOLECULAR PLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994)(enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments).

(L) A gene encoding a virus-specific antibody. See, for example,Tavladoraki, et al., (1993) Nature 366:469, who show that transgenicplants expressing recombinant antibody genes are protected from virusattack.

(M) A polynucleotide encoding a developmental-arrestive protein producedin nature by a pathogen or a parasite. Thus, fungal endoalpha-1,4-D-polygalacturonases facilitate fungal colonization and plantnutrient release by solubilizing plant cell wallhomo-alpha-1,4-D-galacturonase. See, Lamb, et al., (1992) Bio/Technology10:1436. The cloning and characterization of a gene which encodes a beanendopolygalacturonase-inhibiting protein is described by Toubart, etal., (1992) Plant J. 2:367.

(N) A polynucleotide encoding a developmental-arrestive protein producedin nature by a plant. For example, Logemann, et al., (1992)Bio/Technology 10:305, have shown that transgenic plants expressing thebarley ribosome-inactivating gene have an increased resistance to fungaldisease.

(O) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, (1995) Current Biology5(2), Pieterse and Van Loon, (2004) Curr. Opin. Plant Bio. 7(4):456-64and Somssich, (2003) Cell 113(7):815-6.

(P) Antifungal genes (Cornelissen and Melchers, (1993) Pl. Physiol.101:709-712 and Parijs, et al., (1991) Planta 183:258-264 and Bushnell,et al., (1998) Can. J. of Plant Path. 20(2):137-149. Also see, U.S.patent application Ser. Nos. 09/950,933; 11/619,645; 11/657,710;11/748,994; 11/774,121 and U.S. Pat. Nos. 6,891,085 and 7,306,946. LysMReceptor-like kinases for the perception of chitin fragments as a firststep in plant defense response against fungal pathogens (US2012/0110696).

(Q) Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see, U.S. Pat. Nos. 5,716,820; 5,792,931; 5,798,255;5,846,812; 6,083,736; 6,538,177; 6,388,171 and 6,812,380.

(R) A polynucleotide encoding a Cystatin and cysteine proteinaseinhibitors. See, U.S. Pat. No. 7,205,453.

(S) Defensin genes. See, WO 2003/000863 and U.S. Pat. Nos. 6,911,577;6,855,865; 6,777,592 and 7,238,781.

(T) Genes conferring resistance to nematodes. See, e.g., PCT ApplicationWO 1996/30517; PCT Application WO 1993/19181, WO 2003/033651 and Urwin,et al., (1998) Planta 204:472-479, Williamson, (1999) Curr Opin PlantBio. 2(4):327-31; U.S. Pat. Nos. 6,284,948 and 7,301,069 and miR164genes (WO 2012/058266).

(U) Genes that confer resistance to Phytophthora Root Rot, such as theRps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps genes.See, for example, Shoemaker, et al., Phytophthora Root Rot ResistanceGene Mapping in Soybean, Plant Genome IV Conference, San Diego, Calif.(1995).

(V) Genes that confer resistance to Brown Stem Rot, such as described inU.S. Pat. No. 5,689,035 and incorporated by reference for this purpose.

(W) Genes that confer resistance to Colletotrichum, such as described inUS Patent Application Publication US 2009/0035765 and incorporated byreference for this purpose. This includes the Rcg locus that may beutilized as a single locus conversion.

2. Transgenes that Confer Resistance to a Herbicide, for Example:

(A) A polynucleotide encoding resistance to a herbicide that inhibitsthe growing point or meristem, such as an imidazolinone or asulfonylurea. Exemplary genes in this category code for mutant ALS andAHAS enzyme as described, for example, by Lee, et al., (1988) EMBO J.7:1241 and Miki, et al., (1990) Theor. Appl. Genet. 80:449,respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870;5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937 and5,378,824; U.S. patent application Ser. No. 11/683,737 and InternationalPublication WO 1996/33270.

(B) A polynucleotide encoding a protein for resistance to Glyphosate(resistance imparted by mutant 5-enolpyruvl-3-phosphikimate synthase(EPSP) and aroA genes, respectively) and other phosphono compounds suchas glufosinate (phosphinothricin acetyl transferase (PAT) andStreptomyces hygroscopicus phosphinothricin acetyl transferase (bar)genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones(ACCase inhibitor-encoding genes). See, for example, U.S. Pat. No.4,940,835 to Shah, et al., which discloses the nucleotide sequence of aform of EPSPS which can confer glyphosate resistance. U.S. Pat. No.5,627,061 to Barry, et al., also describes genes encoding EPSPS enzymes.See also, U.S. Pat. Nos. 6,566,587; 6,338,961; 6,248,876; 6,040,497;5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642;5,094,945, 4,940,835; 5,866,775; 6,225,114; 6,130,366; 5,310,667;4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E and5,491,288 and International Publications EP 1173580; WO 2001/66704; EP1173581 and EP 1173582, which are incorporated herein by reference forthis purpose. Glyphosate resistance is also imparted to plants thatexpress a gene encoding a glyphosate oxido-reductase enzyme as describedmore fully in U.S. Pat. Nos. 5,776,760 and 5,463,175, which areincorporated herein by reference for this purpose. In addition,glyphosate resistance can be imparted to plants by the over expressionof genes encoding glyphosate N-acetyltransferase. See, for example, U.S.Pat. Nos. 7,462,481; 7,405,074 and US Patent Application PublicationNumber US 2008/0234130. A DNA molecule encoding a mutant aroA gene canbe obtained under ATCC® Accession Number 39256, and the nucleotidesequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 toComai. EP Application Number 0 333 033 to Kumada, et al., and U.S. Pat.No. 4,975,374 to Goodman, et al., disclose nucleotide sequences ofglutamine synthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in EP ApplicationNumbers 0 242 246 and 0 242 236 to Leemans, et al., De Greef, et al.,(1989) Bio/Technology 7:61, describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. See also, U.S. Pat. Nos. 5,969,213; 5,489,520;5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024;6,177,616 and 5,879,903, which are incorporated herein by reference forthis purpose. Exemplary genes conferring resistance to phenoxyproprionic acids and cyclohexones, such as sethoxydim and haloxyfop, arethe Acc1-S1, Acc1-S2 and Acc1-S3 genes described by Marshall, et al.,(1992) Theor. Appl. Genet. 83:435.

(C) A polynucleotide encoding a protein for resistance to herbicide thatinhibits photosynthesis, such as a triazine (psbA and gs+ genes) and abenzonitrile (nitrilase gene). Przibilla, et al., (1991) Plant Cell3:169, describe the transformation of Chlamydomonas with plasmidsencoding mutant psbA genes. Nucleotide sequences for nitrilase genes aredisclosed in U.S. Pat. No. 4,810,648 to Stalker and DNA moleculescontaining these genes are available under ATCC® Accession Numbers53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes, et al., (1992) Biochem.J. 285:173.

(D) A polynucleotide encoding a protein for resistance to Acetohydroxyacid synthase, which has been found to make plants that express thisenzyme resistant to multiple types of herbicides, has been introducedinto a variety of plants (see, e.g., Hattori, et al., (1995) Mol GenGenet. 246:419). Other genes that confer resistance to herbicidesinclude: a gene encoding a chimeric protein of rat cytochrome P4507A1and yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., (1994)Plant Physiol 106:17), genes for glutathione reductase and superoxidedismutase (Aono, et al., (1995) Plant Cell Physiol 36:1687) and genesfor various phosphotransferases (Datta, et al., (1992) Plant Mol Biol20:619).

(E) A polynucleotide encoding resistance to a herbicide targetingProtoporphyrinogen oxidase (protox) which is necessary for theproduction of chlorophyll. The protox enzyme serves as the target for avariety of herbicidal compounds. These herbicides also inhibit growth ofall the different species of plants present, causing their totaldestruction. The development of plants containing altered protoxactivity which are resistant to these herbicides are described in U.S.Pat. Nos. 6,288,306; 6,282,83 and 5,767,373 and InternationalPublication WO 2001/12825.

(F) The aad-1 gene (originally from Sphingobium herbicidovorans) encodesthe aryloxyalkanoate dioxygenase (AAD-1) protein. The trait conferstolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate(commonly referred to as “fop” herbicides such as quizalofop)herbicides. The aad-1 gene, itself, for herbicide tolerance in plantswas first disclosed in WO 2005/107437 (see also, US 2009/0093366). Theaad-12 gene, derived from Delftia acidovorans, which encodes thearyloxyalkanoate dioxygenase (AAD-12) protein that confers tolerance to2,4-dichlorophenoxyacetic acid and pyridyloxyacetate herbicides bydeactivating several herbicides with an aryloxyalkanoate moiety,including phenoxy auxin (e.g., 2,4-D, MCPA), as well as pyridyloxyauxins (e.g., fluroxypyr, triclopyr).

(G) A polynucleotide encoding a herbicide resistant dicambamonooxygenase disclosed in US Patent Application Publication2003/0135879 for imparting dicamba tolerance;

(H) A polynucleotide molecule encoding bromoxynil nitrilase (Bxn)disclosed in U.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance;

(I) A polynucleotide molecule encoding phytoene (crtl) described inMisawa, et al., (1993) Plant J. 4:833-840 and in Misawa, et al., (1994)Plant J. 6:481-489 for norflurazon tolerance.

3. Transgenes that Confer or Contribute to an Altered GrainCharacteristic

Such as:

(A) Altered fatty acids, for example, by

(1) Down-regulation of stearoyl-ACP to increase stearic acid content ofthe plant. See, Knultzon, et al., (1992) Proc. Natl. Acad. Sci. USA89:2624 and WO 1999/64579 (Genes to Alter Lipid Profiles in Corn).

(2) Elevating oleic acid via FAD-2 gene modification and/or decreasinglinolenic acid via FAD-3 gene modification (see, U.S. Pat. Nos.6,063,947; 6,323,392; 6,372,965 and WO 1993/11245).

(3) Altering conjugated linolenic or linoleic acid content, such as inWO 2001/12800.

(4) Altering LEC1, AGP, Dek1, Superal1, mi1 ps, and various Ipa genessuch as Ipa1, Ipa3, hpt or hggt. For example, see, WO 2002/42424, WO1998/22604, WO 2003/011015, WO 2002/057439, WO 2003/011015, U.S. Pat.Nos. 6,423,886, 6,197,561, 6,825,397 and US Patent ApplicationPublication Numbers US 2003/0079247, US 2003/0204870 and Rivera-Madrid,et al., (1995) Proc. Natl. Acad. Sci. 92:5620-5624.

(5) Genes encoding delta-8 desaturase for making long-chainpolyunsaturated fatty acids (U.S. Pat. Nos. 8,058,571 and 8,338,152),delta-9 desaturase for lowering saturated fats (U.S. Pat. No.8,063,269), Primula Δ6-desaturase for improving omega-3 fatty acidprofiles.

(6) Isolated nucleic acids and proteins associated with lipid and sugarmetabolism regulation, in particular, lipid metabolism protein (LMP)used in methods of producing transgenic plants and modulating levels ofseed storage compounds including lipids, fatty acids, starches or seedstorage proteins and use in methods of modulating the seed size, seednumber, seed weights, root length and leaf size of plants (EP 2404499).

(7) Altering expression of a High-Level Expression of Sugar-Inducible 2(HSI2) protein in the plant to increase or decrease expression of HSI2in the plant. Increasing expression of HSI2 increases oil content whiledecreasing expression of HSI2 decreases abscisic acid sensitivity and/orincreases drought resistance (US Patent Application Publication Number2012/0066794).

(8) Expression of cytochrome b5 (Cb5) alone or with FAD2 to modulate oilcontent in plant seed, particularly to increase the levels of omega-3fatty acids and improve the ratio of omega-6 to omega-3 fatty acids (USPatent Application Publication Number 2011/0191904).

(9) Nucleic acid molecules encoding wrinkled1-like polypeptides formodulating sugar metabolism (U.S. Pat. No. 8,217,223).

(B) Altered phosphorus content, for example, by the

(1) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant. Forexample, see, Van Hartingsveldt, et al., (1993) Gene 127:87, for adisclosure of the nucleotide sequence of an Aspergillus niger phytasegene.

(2) Modulating a gene that reduces phytate content. In maize, this, forexample, could be accomplished, by cloning and then re-introducing DNAassociated with one or more of the alleles, such as the LPA alleles,identified in maize mutants characterized by low levels of phytic acid,such as in WO 2005/113778 and/or by altering inositol kinase activity asin WO 2002/059324, US Patent Application Publication Number2003/0009011, WO 2003/027243, US Patent Application Publication Number2003/0079247, WO 1999/05298, U.S. Pat. Nos. 6,197,561, 6,291,224,6,391,348, WO 2002/059324, US Patent Application Publication Number2003/0079247, WO 1998/45448, WO 1999/55882, WO 2001/04147.

(C) Altered carbohydrates affected, for example, by altering a gene foran enzyme that affects the branching pattern of starch or, a genealtering thioredoxin such as NTR and/or TRX (see, U.S. Pat. No.6,531,648, which is incorporated by reference for this purpose) and/or agamma zein knock out or mutant such as cs27 or TUSC27 or en27 (see, U.S.Pat. No. 6,858,778 and US Patent Application Publication Number2005/0160488, US Patent Application Publication Number 2005/0204418,which are incorporated by reference for this purpose). See, Shiroza, etal., (1988) J. Bacteriol. 170:810 (nucleotide sequence of Streptococcusmutant fructosyltransferase gene), Steinmetz, et al., (1985) Mol. Gen.Genet. 200:220 (nucleotide sequence of Bacillus subtilis levansucrasegene), Pen, et al., (1992) Bio/Technology 10:292 (production oftransgenic plants that express Bacillus licheniformis alpha-amylase),Elliot, et al., (1993) Plant Molec. Biol. 21:515 (nucleotide sequencesof tomato invertase genes), Søgaard, et al., (1993) J. Biol. Chem.268:22480 (site-directed mutagenesis of barley alpha-amylase gene) andFisher, et al., (1993) Plant Physiol. 102:1045 (maize endosperm starchbranching enzyme II), WO 1999/10498 (improved digestibility and/orstarch extraction through modification of UDP-D-xylose 4-epimerase,Fragile 1 and 2, Ref1, HCHL, C4H), U.S. Pat. No. 6,232,529 (method ofproducing high oil seed by modification of starch levels (AGP)). Thefatty acid modification genes mentioned herein may also be used toaffect starch content and/or composition through the interrelationshipof the starch and oil pathways.

(D) Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, see, U.S. Pat. No. 6,787,683,US Patent Application Publication Number 2004/0034886 and WO 2000/68393involving the manipulation of antioxidant levels and WO 2003/082899through alteration of a homogentisate geranyl geranyl transferase(hggt).

(E) Altered essential seed amino acids. For example, see, U.S. Pat. No.6,127,600 (method of increasing accumulation of essential amino acids inseeds), U.S. Pat. No. 6,080,913 (binary methods of increasingaccumulation of essential amino acids in seeds), U.S. Pat. No. 5,990,389(high lysine), WO 1999/40209 (alteration of amino acid compositions inseeds), WO 1999/29882 (methods for altering amino acid content ofproteins), U.S. Pat. No. 5,850,016 (alteration of amino acidcompositions in seeds), WO 1998/20133 (proteins with enhanced levels ofessential amino acids), U.S. Pat. No. 5,885,802 (high methionine), U.S.Pat. No. 5,885,801 (high threonine), U.S. Pat. No. 6,664,445 (plantamino acid biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increasedlysine and threonine), U.S. Pat. No. 6,441,274 (plant tryptophansynthase beta subunit), U.S. Pat. No. 6,346,403 (methionine metabolicenzymes), U.S. Pat. No. 5,939,599 (high sulfur), U.S. Pat. No. 5,912,414(increased methionine), WO 1998/56935 (plant amino acid biosyntheticenzymes), WO 1998/45458 (engineered seed protein having higherpercentage of essential amino acids), WO 1998/42831 (increased lysine),U.S. Pat. No. 5,633,436 (increasing sulfur amino acid content), U.S.Pat. No. 5,559,223 (synthetic storage proteins with defined structurecontaining programmable levels of essential amino acids for improvementof the nutritional value of plants), WO 1996/01905 (increasedthreonine), WO 1995/15392 (increased lysine), US Patent ApplicationPublication Number 2003/0163838, US Patent Application PublicationNumber 2003/0150014, US Patent Application Publication Number2004/0068767, U.S. Pat. No. 6,803,498, WO 2001/79516.

4. Genes that Control Male-Sterility:

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar, et al., and chromosomal translocationsas described by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen, et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

(A) Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN—Ac-PPT (WO 2001/29237).

(B) Introduction of various stamen-specific promoters (WO 1992/13956, WO1992/13957).

(C) Introduction of the barnase and the barstar gene (Paul, et al.,(1992) Plant Mol. Biol. 19:611-622).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341; 6,297,426; 5,478,369;5,824,524; 5,850,014 and 6,265,640, all of which are hereby incorporatedby reference.

5. Genes that Create a Site for Site Specific DNA Integration.

This includes the introduction of FRT sites that may be used in theFLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system.For example, see, Lyznik, et al., (2003) Plant Cell Rep 21:925-932 andWO 1999/25821, which are hereby incorporated by reference. Other systemsthat may be used include the Gin recombinase of phage Mu (Maeser, etal., (1991) Vicki Chandler, The Maize Handbook ch. 118 (Springer-Verlag1994), the Pin recombinase of E. coli (Enomoto, et al., 1983) and theR/RS system of the pSRi plasmid (Araki, et al., 1992).

6. Genes that Affect Abiotic Stress Resistance

Including but not limited to flowering, ear and seed development,enhancement of nitrogen utilization efficiency, altered nitrogenresponsiveness, drought resistance or tolerance, cold resistance ortolerance and salt resistance or tolerance and increased yield understress.

(A) For example, see: WO 2000/73475 where water use efficiency isaltered through alteration of malate; U.S. Pat. Nos. 5,892,009,5,965,705, 5,929,305, 5,891,859, 6,417,428, 6,664,446, 6,706,866,6,717,034, 6,801,104, WO 2000/060089, WO 2001/026459, WO 2001/035725, WO2001/034726, WO 2001/035727, WO 2001/036444, WO 2001/036597, WO2001/036598, WO 2002/015675, WO 2002/017430, WO 2002/077185, WO2002/079403, WO 2003/013227, WO 2003/013228, WO 2003/014327, WO2004/031349, WO 2004/076638, WO 199809521.

(B) WO 199938977 describing genes, including CBF genes and transcriptionfactors effective in mitigating the negative effects of freezing, highsalinity and drought on plants, as well as conferring other positiveeffects on plant phenotype.

(C) US Patent Application Publication Number 2004/0148654 and WO2001/36596 where abscisic acid is altered in plants resulting inimproved plant phenotype such as increased yield and/or increasedtolerance to abiotic stress.

(D) WO 2000/006341, WO 2004/090143, U.S. Pat. Nos. 7,531,723 and6,992,237 where cytokinin expression is modified resulting in plantswith increased stress tolerance, such as drought tolerance, and/orincreased yield. Also see, WO 2002/02776, WO 2003/052063, JP2002/281975, U.S. Pat. No. 6,084,153, WO 2001/64898, U.S. Pat. Nos.6,177,275 and 6,107,547 (enhancement of nitrogen utilization and alterednitrogen responsiveness).

(E) For ethylene alteration, see, US Patent Application PublicationNumber 2004/0128719, US Patent Application Publication Number2003/0166197 and WO 2000/32761.

(F) For plant transcription factors or transcriptional regulators ofabiotic stress, see, e.g., US Patent Application Publication Number2004/0098764 or US Patent Application Publication Number 2004/0078852.

(G) Genes that increase expression of vacuolar pyrophosphatase such asAVP1 (U.S. Pat. No. 8,058,515) for increased yield; nucleic acidencoding a HSFA4 or a HSFA5 (Heat Shock Factor of the class A4 or A5)polypeptides, an oligopeptide transporter protein (OPT4-like)polypeptide; a plastochron2-like (PLA2-like) polypeptide or a Wuschelrelated homeobox 1-like (WOX1-like) polypeptide (U. Patent ApplicationPublication Number US 2011/0283420).

(H) Down regulation of polynucleotides encoding poly (ADP-ribose)polymerase (PARP) proteins to modulate programmed cell death (U.S. Pat.No. 8,058,510) for increased vigor.

(I) Polynucleotide encoding DTP21 polypeptides for conferring droughtresistance (US Patent Application Publication Number US 2011/0277181).

(J) Nucleotide sequences encoding ACC Synthase 3 (ACS3) proteins formodulating development, modulating response to stress, and modulatingstress tolerance (US Patent Application Publication Number US2010/0287669).

(K) Polynucleotides that encode proteins that confer a drought tolerancephenotype (DTP) for conferring drought resistance (WO 2012/058528).

(L) Tocopherol cyclase (TC) genes for conferring drought and salttolerance (US Patent Application Publication Number 2012/0272352).

(M) CAAX amino terminal family proteins for stress tolerance (U.S. Pat.No. 8,338,661).

(N) Mutations in the SAL1 encoding gene have increased stress tolerance,including increased drought resistant (US Patent Application PublicationNumber 2010/0257633).

(O) Expression of a nucleic acid sequence encoding a polypeptideselected from the group consisting of: GRF polypeptide, RAA1-likepolypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptideincreasing yield-related traits (US Patent Application PublicationNumber 2011/0061133).

(P) Modulating expression in a plant of a nucleic acid encoding a ClassIII Trehalose Phosphate Phosphatase (TPP) polypeptide for enhancingyield-related traits in plants, particularly increasing seed yield (USPatent Application Publication Number 2010/0024067).

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants, see e.g., WO1997/49811 (LHY), WO 1998/56918 (ESD4), WO 1997/10339 and U.S. Pat. No.6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO 1996/14414 (CON), WO1996/38560, WO 2001/21822 (VRN1), WO 2000/44918 (VRN2), WO 1999/49064(GI), WO 2000/46358 (FR1), WO 1997/29123, U.S. Pat. Nos. 6,794,560,6,307,126 (GAI), WO 1999/09174 (D8 and Rht) and WO 2004/076638 and WO2004/031349 (transcription factors).

7. Genes that Confer Increased Yield

(A) A transgenic crop plant transformed by a1-AminoCyclopropane-1-Carboxylate Deaminase-like Polypeptide (ACCDP)coding nucleic acid, wherein expression of the nucleic acid sequence inthe crop plant results in the plant's increased root growth, and/orincreased yield, and/or increased tolerance to environmental stress ascompared to a wild type variety of the plant (U.S. Pat. No. 8,097,769).

(B) Over-expression of maize zinc finger protein gene (Zm-ZFP1) using aseed preferred promoter has been shown to enhance plant growth, increasekernel number and total kernel weight per plant (US Patent ApplicationPublication Number 2012/0079623).

(C) Constitutive over-expression of maize lateral organ boundaries (LOB)domain protein (Zm-LOBDP1) has been shown to increase kernel number andtotal kernel weight per plant (US Patent Application Publication Number2012/0079622).

(D) Enhancing yield-related traits in plants by modulating expression ina plant of a nucleic acid encoding a VIM1 (Variant in Methylation1)-like polypeptide or a VTC2-like (GDP-L-galactose phosphorylase)polypeptide or a DUF1685 polypeptide or an ARF6-like (Auxin ResponsiveFactor) polypeptide (WO 2012/038893).

(E) Modulating expression in a plant of a nucleic acid encoding aSte20-like polypeptide or a homologue thereof gives plants havingincreased yield relative to control plants (EP 2431472).

(F) Genes encoding nucleoside diphosphatase kinase (NDK) polypeptidesand homologs thereof for modifying the plant's root architecture (USPatent Application Publication Number 2009/0064373).

8. Genes that Confer Plant Digestibility.

(A) Altering the level of xylan present in the cell wall of a plant bymodulating expression of xylan synthase (U.S. Pat. No. 8,173,866).

In some embodiment the stacked trait may be a trait or event that hasreceived regulatory approval including but not limited to the eventswith regulatory approval that are well known to one skilled in the artand can be found at the Center for Environmental Risk Assessment(cera-gmc.org/?action=gm_crop_database, which can be accessed using thewww prefix) and at the International Service for the Acquisition ofAgri-Biotech Applications (isaaa.org/gmapprovaldatabase/default.asp,which can be accessed using the www prefix).

Gene Silencing

In some embodiments the stacked trait may be in the form of silencing ofone or more polynucleotides of interest resulting in suppression of oneor more target pest polypeptides. In some embodiments the silencing isachieved through the use of a suppression DNA construct.

In some embodiments one or more polynucleotide encoding the polypeptidesof the IPD090 polypeptide or fragments or variants thereof may bestacked with one or more polynucleotides encoding one or morepolypeptides having insecticidal activity or agronomic traits as setforth supra and optionally may further include one or morepolynucleotides providing for gene silencing of one or more targetpolynucleotides as discussed infra.

“Suppression DNA construct” is a recombinant DNA construct which whentransformed or stably integrated into the genome of the plant, resultsin “silencing” of a target gene in the plant. The target gene may beendogenous or transgenic to the plant. “Silencing,” as used herein withrespect to the target gene, refers generally to the suppression oflevels of mRNA or protein/enzyme expressed by the target gene, and/orthe level of the enzyme activity or protein functionality. The term“suppression” includes lower, reduce, decline, decrease, inhibit,eliminate and prevent. “Silencing” or “gene silencing” does not specifymechanism and is inclusive, and not limited to, anti-sense,cosuppression, viral-suppression, hairpin suppression, stem-loopsuppression, RNAi-based approaches and small RNA-based approaches.

A suppression DNA construct may comprise a region derived from a targetgene of interest and may comprise all or part of the nucleic acidsequence of the sense strand (or antisense strand) of the target gene ofinterest. Depending upon the approach to be utilized, the region may be100% identical or less than 100% identical (e.g., at least 50% or anyinteger between 51% and 100% identical) to all or part of the sensestrand (or antisense strand) of the gene of interest.

Suppression DNA constructs are well-known in the art, are readilyconstructed once the target gene of interest is selected, and include,without limitation, cosuppression constructs, antisense constructs,viral-suppression constructs, hairpin suppression constructs, stem-loopsuppression constructs, double-stranded RNA-producing constructs, andmore generally, RNAi (RNA interference) constructs and small RNAconstructs such as siRNA (short interfering RNA) constructs and miRNA(microRNA) constructs.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.

“Antisense RNA” refers to an RNA transcript that is complementary to allor part of a target primary transcript or mRNA and that blocks theexpression of a target isolated nucleic acid fragment. Thecomplementarity of an antisense RNA may be with any part of the specificgene transcript, i.e., at the 5′ non-coding sequence, 3′ non-codingsequence, introns or the coding sequence.

“Cosuppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of the target protein. “Sense” RNArefers to RNA transcript that includes the mRNA and can be translatedinto protein within a cell or in vitro. Cosuppression constructs inplants have been previously designed by focusing on overexpression of anucleic acid sequence having homology to a native mRNA, in the senseorientation, which results in the reduction of all RNA having homologyto the overexpressed sequence (see, Vaucheret, et al., (1998) Plant J.16:651-659 and Gura, (2000) Nature 404:804-808).

Another variation describes the use of plant viral sequences to directthe suppression of proximal mRNA encoding sequences (PCT Publication WO1998/36083).

Recent work has described the use of “hairpin” structures thatincorporate all or part, of an mRNA encoding sequence in a complementaryorientation that results in a potential “stem-loop” structure for theexpressed RNA (PCT Publication WO 1999/53050). In this case the stem isformed by polynucleotides corresponding to the gene of interest insertedin either sense or anti-sense orientation with respect to the promoterand the loop is formed by some polynucleotides of the gene of interest,which do not have a complement in the construct. This increases thefrequency of cosuppression or silencing in the recovered transgenicplants. For review of hairpin suppression, see, Wesley, et al., (2003)Methods in Molecular Biology, Plant Functional Genomics: Methods andProtocols 236:273-286.

A construct where the stem is formed by at least 30 nucleotides from agene to be suppressed and the loop is formed by a random nucleotidesequence has also effectively been used for suppression (PCT PublicationWO 1999/61632).

The use of poly-T and poly-A sequences to generate the stem in thestem-loop structure has also been described (PCT Publication WO2002/00894).

Yet another variation includes using synthetic repeats to promoteformation of a stem in the stem-loop structure. Transgenic organismsprepared with such recombinant DNA fragments have been shown to havereduced levels of the protein encoded by the nucleotide fragment formingthe loop as described in PCT Publication WO 2002/00904.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire, et al., (1998) Nature 391:806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing (PTGS) or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire, et al., (1999) TrendsGenet. 15:358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA of viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response through amechanism that has yet to be fully characterized.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein, et al., (2001) Nature 409:363).Short interfering RNAs derived from dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes (Elbashir, et aL, (2001) Genes Dev. 15:188). Dicer has alsobeen implicated in the excision of 21- and 22-nucleotide small temporalRNAs (stRNAs) from precursor RNA of conserved structure that areimplicated in translational control (Hutvagner, et al., (2001) Science293:834). The RNAi response also features an endonuclease complex,commonly referred to as an RNA-induced silencing complex (RISC), whichmediates cleavage of single-stranded RNA having sequence complementarityto the antisense strand of the siRNA duplex. Cleavage of the target RNAtakes place in the middle of the region complementary to the antisensestrand of the siRNA duplex (Elbashir, et al., (2001) Genes Dev. 15:188).In addition, RNA interference can also involve small RNA (e.g., miRNA)mediated gene silencing, presumably through cellular mechanisms thatregulate chromatin structure and thereby prevent transcription of targetgene sequences (see, e.g., Allshire, (2002) Science 297:1818-1819;Volpe, et al., (2002) Science 297:1833-1837; Jenuwein, (2002) Science297:2215-2218 and Hall, et al., (2002) Science 297:2232-2237). As such,miRNA molecules of the disclosure can be used to mediate gene silencingvia interaction with RNA transcripts or alternately by interaction withparticular gene sequences, wherein such interaction results in genesilencing either at the transcriptional or post-transcriptional level.

Methods and compositions are further provided which allow for anincrease in RNAi produced from the silencing element. In suchembodiments, the methods and compositions employ a first polynucleotidecomprising a silencing element for a target pest sequence operablylinked to a promoter active in the plant cell; and, a secondpolynucleotide comprising a suppressor enhancer element comprising thetarget pest sequence or an active variant or fragment thereof operablylinked to a promoter active in the plant cell. The combined expressionof the silencing element with suppressor enhancer element leads to anincreased amplification of the inhibitory RNA produced from thesilencing element over that achievable with only the expression of thesilencing element alone. In addition to the increased amplification ofthe specific RNAi species itself, the methods and compositions furtherallow for the production of a diverse population of RNAi species thatcan enhance the effectiveness of disrupting target gene expression. Assuch, when the suppressor enhancer element is expressed in a plant cellin combination with the silencing element, the methods and compositioncan allow for the systemic production of RNAi throughout the plant; theproduction of greater amounts of RNAi than would be observed with justthe silencing element construct alone; and, the improved loading of RNAiinto the phloem of the plant, thus providing better control of phloemfeeding insects by an RNAi approach. Thus, the various methods andcompositions provide improved methods for the delivery of inhibitory RNAto the target organism. See, for example, US Patent ApplicationPublication 2009/0188008.

As used herein, a “suppressor enhancer element” comprises apolynucleotide comprising the target sequence to be suppressed or anactive fragment or variant thereof. It is recognized that the suppressorenhancer element need not be identical to the target sequence, butrather, the suppressor enhancer element can comprise a variant of thetarget sequence, so long as the suppressor enhancer element hassufficient sequence identity to the target sequence to allow for anincreased level of the RNAi produced by the silencing element over thatachievable with only the expression of the silencing element. Similarly,the suppressor enhancer element can comprise a fragment of the targetsequence, wherein the fragment is of sufficient length to allow for anincreased level of the RNAi produced by the silencing element over thatachievable with only the expression of the silencing element.

It is recognized that multiple suppressor enhancer elements from thesame target sequence or from different target sequences or fromdifferent regions of the same target sequence can be employed. Forexample, the suppressor enhancer elements employed can comprisefragments of the target sequence derived from different region of thetarget sequence (i.e., from the 3′UTR, coding sequence, intron, and/or5′UTR). Further, the suppressor enhancer element can be contained in anexpression cassette, as described elsewhere herein, and in specificembodiments, the suppressor enhancer element is on the same or on adifferent DNA vector or construct as the silencing element. Thesuppressor enhancer element can be operably linked to a promoter asdisclosed herein. It is recognized that the suppressor enhancer elementcan be expressed constitutively or alternatively, it may be produced ina stage-specific manner employing the various inducible ortissue-preferred or developmentally regulated promoters that arediscussed elsewhere herein.

In specific embodiments, employing both a silencing element and thesuppressor enhancer element the systemic production of RNAi occursthroughout the entire plant. In further embodiments, the plant or plantparts of the disclosure have an improved loading of RNAi into the phloemof the plant than would be observed with the expression of the silencingelement construct alone and, thus provide better control of phloemfeeding insects by an RNAi approach. In specific embodiments, theplants, plant parts and plant cells of the disclosure can further becharacterized as allowing for the production of a diversity of RNAispecies that can enhance the effectiveness of disrupting target geneexpression.

In specific embodiments, the combined expression of the silencingelement and the suppressor enhancer element increases the concentrationof the inhibitory RNA in the plant cell, plant, plant part, plant tissueor phloem over the level that is achieved when the silencing element isexpressed alone.

As used herein, an “increased level of inhibitory RNA” comprises anystatistically significant increase in the level of RNAi produced in aplant having the combined expression when compared to an appropriatecontrol plant. For example, an increase in the level of RNAi in theplant, plant part or the plant cell can comprise at least about a 1%,about a 1%-5%, about a 5%-10%, about a 10%-20%, about a 20%-30%, about a30%-40%, about a 40%-50%, about a 50%-60%, about 60-70%, about 70%-80%,about a 80%-90%, about a 90%-100% or greater increase in the level ofRNAi in the plant, plant part, plant cell or phloem when compared to anappropriate control. In other embodiments, the increase in the level ofRNAi in the plant, plant part, plant cell or phloem can comprise atleast about a 1 fold, about a 1 fold-5 fold, about a 5 fold-10 fold,about a 10 fold-20 fold, about a 20 fold-30 fold, about a 30 fold-40fold, about a 40 fold-50 fold, about a 50 fold-60 fold, about 60 fold-70fold, about 70 fold-80 fold, about a 80 fold-90 fold, about a 90fold-100 fold or greater increase in the level of RNAi in the plant,plant part, plant cell or phloem when compared to an appropriatecontrol. Examples of combined expression of the silencing element withsuppressor enhancer element for the control of Stinkbugs and Lygus canbe found in US Patent Application Publication 2011/0301223 and US PatentApplication Publication 2009/0192117.

Some embodiments relate to down-regulation of expression of target genesin insect pest species by interfering ribonucleic acid (RNA) molecules.PCT Publication WO 2007/074405 describes methods of inhibitingexpression of target genes in invertebrate pests including Coloradopotato beetle. PCT Publication WO 2005/110068 describes methods ofinhibiting expression of target genes in invertebrate pests including inparticular Western corn rootworm as a means to control insectinfestation. Furthermore, PCT Publication WO 2009/091864 describescompositions and methods for the suppression of target genes from insectpest species including pests from the Lygus genus. Nucleic acidmolecules including RNAi for targeting the vacuolar ATPase H subunit,useful for controlling a coleopteran pest population and infestation asdescribed in US Patent Application Publication 2012/0198586. PCTPublication WO 2012/055982 describes ribonucleic acid (RNA or doublestranded RNA) that inhibits or down regulates the expression of a targetgene that encodes: an insect ribosomal protein such as the ribosomalprotein L19, the ribosomal protein L40 or the ribosomal protein S27A; aninsect proteasome subunit such as the Rpn6 protein, the Pros 25, theRpn2 protein, the proteasome beta 1 subunit protein or the Pros beta 2protein; an insect β-coatomer of the COPI vesicle, the γ-coatomer of theCOPI vesicle, the β′-coatomer protein or the ζ-coatomer of the COPIvesicle; an insect Tetraspanine 2 A protein which is a putativetransmembrane domain protein; an insect protein belonging to the actinfamily such as Actin 5C; an insect ubiquitin-5E protein; an insect Sec23protein which is a GTPase activator involved in intracellular proteintransport; an insect crinkled protein which is an unconventional myosinwhich is involved in motor activity; an insect crooked neck proteinwhich is involved in the regulation of nuclear alternative mRNAsplicing; an insect vacuolar H+-ATPase G-subunit protein and an insectTbp-1 such as Tat-binding protein. PCT publication WO 2007/035650describes ribonucleic acid (RNA or double stranded RNA) that inhibits ordown regulates the expression of a target gene that encodes Snf7. USPatent Application publication 20150176009 describes polynucleotidesilencing elements targeting Rnapii-140 that confer resistance tocoleopteran pests. US Patent Application publication 2011/0054007describes polynucleotide silencing elements targeting RPS10. US PatentApplication publication 2014/0275208 and US2015/0257389 describespolynucleotide silencing elements targeting RyanR and PAT3. US PatentApplication Publications 2012/029750, US 20120297501, and 2012/0322660describe interfering ribonucleic acids (RNA or double stranded RNA) thatfunctions upon uptake by an insect pest species to down-regulateexpression of a target gene in said insect pest, wherein the RNAcomprises at least one silencing element wherein the silencing elementis a region of double-stranded RNA comprising annealed complementarystrands, one strand of which comprises or consists of a sequence ofnucleotides which is at least partially complementary to a targetnucleotide sequence within the target gene. US Patent ApplicationPublication 2012/0164205 describe potential targets for interferingdouble stranded ribonucleic acids for inhibiting invertebrate pestsincluding: a Chd3 Homologous Sequence, a Beta-Tubulin HomologousSequence, a 40 kDa V-ATPase Homologous Sequence, a EF1α HomologousSequence, a 26S Proteosome Subunit p28 Homologous Sequence, a JuvenileHormone Epoxide Hydrolase Homologous Sequence, a Swelling DependentChloride Channel Protein Homologous Sequence, a Glucose-6-Phosphate1-Dehydrogenase Protein Homologous Sequence, an Act42A ProteinHomologous Sequence, a ADP-Ribosylation Factor 1 Homologous Sequence, aTranscription Factor IIB Protein Homologous Sequence, a ChitinaseHomologous Sequences, a Ubiquitin Conjugating Enzyme HomologousSequence, a Glyceraldehyde-3-Phosphate Dehydrogenase HomologousSequence, an Ubiquitin B Homologous Sequence, a Juvenile HormoneEsterase Homolog, and an Alpha Tubuliln Homologous Sequence.

Use in Pesticidal Control

General methods for employing strains comprising a nucleic acid sequenceof the embodiments or a variant thereof, in pesticide control or inengineering other organisms as pesticidal agents are known in the art.

Microorganism hosts that are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one ormore crops of interest may be selected. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment with the wild-type microorganisms, provide for stablemaintenance and expression of the gene expressing the IPD090 polypeptideand desirably provide for improved protection of the pesticide fromenvironmental degradation and inactivation.

Alternatively, the IPD090 polypeptide is produced by introducing aheterologous gene into a cellular host. Expression of the heterologousgene results, directly or indirectly, in the intracellular productionand maintenance of the pesticide. These cells are then treated underconditions that prolong the activity of the toxin produced in the cellwhen the cell is applied to the environment of target pest(s). Theresulting product retains the toxicity of the toxin. These naturallyencapsulated IPD090 polypeptides may then be formulated in accordancewith conventional techniques for application to the environment hostinga target pest, e.g., soil, water, and foliage of plants. See, forexample EPA 0192319, and the references cited therein.

Pesticidal Compositions

In some embodiments the active ingredients can be applied in the form ofcompositions and can be applied to the crop area or plant to be treated,simultaneously or in succession, with other compounds. These compoundscan be fertilizers, weed killers, Cryoprotectants, surfactants,detergents, pesticidal soaps, dormant oils, polymers, and/ortime-release or biodegradable carrier formulations that permit long-termdosing of a target area following a single application of theformulation. They can also be selective herbicides, chemicalinsecticides, virucides, microbicides, amoebicides, pesticides,fungicides, bacteriocides, nematocides, molluscicides or mixtures ofseveral of these preparations, if desired, together with furtheragriculturally acceptable carriers, surfactants or application-promotingadjuvants customarily employed in the art of formulation. Suitablecarriers and adjuvants can be solid or liquid and correspond to thesubstances ordinarily employed in formulation technology, e.g. naturalor regenerated mineral substances, solvents, dispersants, wettingagents, tackifiers, binders or fertilizers. Likewise the formulationsmay be prepared into edible “baits” or fashioned into pest “traps” topermit feeding or ingestion by a target pest of the pesticidalformulation.

Methods of applying an active ingredient or an agrochemical compositionthat contains at least one of the IPD090 polypeptide produced by thebacterial strains include leaf application, seed coating and soilapplication. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pest.

The composition may be formulated as a powder, dust, pellet, granule,spray, emulsion, colloid, solution or such like, and may be prepared bysuch conventional means as desiccation, lyophilization, homogenation,extraction, filtration, centrifugation, sedimentation or concentrationof a culture of cells comprising the polypeptide. In all suchcompositions that contain at least one such pesticidal polypeptide, thepolypeptide may be present in a concentration of from about 1% to about99% by weight.

Lepidopteran, Dipteran, Heteropteran, nematode, Hemiptera or Coleopteranpests may be killed or reduced in numbers in a given area by the methodsof the disclosure or may be prophylactically applied to an environmentalarea to prevent infestation by a susceptible pest. Preferably the pestingests or is contacted with, a pesticidally-effective amount of thepolypeptide. “Pesticidally-effective amount” as used herein refers to anamount of the pesticide that is able to bring about death to at leastone pest or to noticeably reduce pest growth, feeding or normalphysiological development. This amount will vary depending on suchfactors as, for example, the specific target pests to be controlled, thespecific environment, location, plant, crop or agricultural site to betreated, the environmental conditions and the method, rate,concentration, stability, and quantity of application of thepesticidally-effective polypeptide composition. The formulations mayalso vary with respect to climatic conditions, environmentalconsiderations, and/or frequency of application and/or severity of pestinfestation.

The pesticide compositions described may be made by formulating eitherthe bacterial cell, Crystal and/or spore suspension or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, desiccated or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial or a suspension in oil (vegetable or mineral) or water oroil/water emulsions or as a wettable powder or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,inert components, dispersants, surfactants, tackifiers, binders, etc.that are ordinarily used in pesticide formulation technology; these arewell known to those skilled in pesticide formulation. The formulationsmay be mixed with one or more solid or liquid adjuvants and prepared byvarious means, e.g., by homogeneously mixing, blending and/or grindingthe pesticidal composition with suitable adjuvants using conventionalformulation techniques. Suitable formulations and application methodsare described in U.S. Pat. No. 6,468,523, herein incorporated byreference. The plants can also be treated with one or more chemicalcompositions, including one or more herbicide, insecticides orfungicides. Exemplary chemical compositions include: Fruits/VegetablesHerbicides: Atrazine, Bromacil, Diuron, Glyphosate, Linuron, Metribuzin,Simazine, Trifluralin, Fluazifop, Glufosinate, Halo sulfuron Gowan,Paraquat, Propyzamide, Sethoxydim, Butafenacil, Halosulfuron,Indaziflam; Fruits/Vegetables Insecticides: Aldicarb, Bacillusthuriengiensis, Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin,Deltamethrin, Diazinon, Malathion, Abamectin,Cyfluthrin/beta-cyfluthrin, Esfenvalerate, Lambda-cyhalothrin,Acequinocyl, Bifenazate, Methoxyfenozide, Novaluron, Chromafenozide,Thiacloprid, Dinotefuran, FluaCrypyrim, Tolfenpyrad, Clothianidin,Spirodiclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr,Cyazypyr, Spinoteram, Triflumuron, Spirotetramat, Imidacloprid,Flubendiamide, Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen,Cyanopyrafen, Imidacloprid, Clothianidin, Thiamethoxam, Spinotoram,Thiodicarb, Flonicamid, Methiocarb, Emamectin-benzoate, Indoxacarb,Forthiazate, Fenamiphos, Cadusaphos, Pyriproxifen, Fenbutatin-oxid,Hexthiazox, Methomyl,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on;Fruits/Vegetables Fungicides: Carbendazim, Chlorothalonil, EBDCs,Sulphur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam,Fosetyl, Iprodione, Kresoxim-methyl, Metalaxyl/mefenoxam,Trifloxystrobin, Ethaboxam, Iprovalicarb, Trifloxystrobin, Fenhexamid,Oxpoconazole fumarate, Cyazofamid, Fenamidone, Zoxamide, Zorvec™,Picoxystrobin, Pyraclostrobin, Cyflufenamid, Boscalid; CerealsHerbicides: Isoproturon, Bromoxynil, Ioxynil, Phenoxies, Chlorsulfuron,Clodinafop, Diclofop, Diflufenican, Fenoxaprop, Florasulam, Fluoroxypyr,Metsulfuron, Triasulfuron, Flucarbazone, Iodosulfuron, Propoxycarbazone,Picolinafen, Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfuron,Thifensulfuron Methyl, Tribenuron, Flupyrsulfuron, Sulfosulfuron,Pyrasulfotole, Pyroxsulam, Flufenacet, Tralkoxydim, Pyroxasulfon;Cereals Fungicides: Carbendazim, Chlorothalonil, Azoxystrobin,Cyproconazole, Cyprodinil, Fenpropimorph, Epoxiconazole,Kresoxim-methyl, Quinoxyfen, Tebuconazole, Trifloxystrobin,Simeconazole, Picoxystrobin, Pyraclostrobin, Dimoxystrobin,Prothioconazole, Fluoxastrobin; Cereals Insecticides: Dimethoate,Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin, β-cyfluthrin,Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid,Acetamiprid, Dinetofuran, Clorphyriphos, Metamidophos,Oxidemethon-methyl, Pirimicarb, Methiocarb; Maize Herbicides: Atrazine,Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-)Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor,Mesotrione, Nicosulfuron, Primisulfuron, Revulin Q®; in Rimsulfuron,Sulcotrione, Foramsulfuron, Topramezone, Tembotrione, Saflufenacil,Thiencarbazone, Flufenacet, Pyroxasulfon; Maize Insecticides:Carbofuran, Chlorpyrifos, Bifenthrin, Fipronil, Imidacloprid,Lambda-Cyhalothrin, Tefluthrin, Terbufos, Thiamethoxam, Clothianidin,Spiromesifen, Flubendiamide, Triflumuron, Rynaxypyr, Deltamethrin,Thiodicarb, β-Cyfluthrin, Cypermethrin, Bifenthrin, Lufenuron,Triflumoron, Tefluthrin, Tebupirimphos, Ethiprole, Cyazypyr,Thiacloprid, Acetamiprid, Dinetofuran, Avermectin, Methiocarb,Spirodiclofen, Spirotetramat; Maize Fungicides: Fenitropan, Thiram,Prothioconazole, Tebuconazole, Trifloxystrobin; Rice Herbicides:Butachlor, Propanil, Azimsulfuron, Bensulfuron, Cyhalofop, Daimuron,Fentrazamide, Imazosulfuron, Mefenacet, Oxaziclomefone, Pyrazosulfuron,Pyributicarb, Quinclorac, Thiobencarb, Indanofan, Flufenacet,Fentrazamide, Halosulfuron, Oxaziclomefone, Benzobicyclon, Pyriftalid,Penoxsulam, Bispyribac, Oxadiargyl, Ethoxysulfuron, Pretilachlor,Mesotrione, Tefuryltrione, Oxadiazone, Fenoxaprop, Pyrimisulfan; RiceInsecticides: Diazinon, Fenitrothion, Fenobucarb, Monocrotophos,Benfuracarb, Buprofezin, Dinotefuran, Fipronil, Imidacloprid,Isoprocarb, Thiacloprid, Chromafenozide, Thiacloprid, Dinotefuran,Clothianidin, Ethiprole, Flubendiamide, Rynaxypyr, Deltamethrin,Acetamiprid, Thiamethoxam, Cyazypyr, Spinosad, Spinotoram,Emamectin-Benzoate, Cypermethrin, Chlorpyriphos, Cartap, Methamidophos,Etofenprox, Triazophos,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Carbofuran, Benfuracarb; Rice Fungicides: Thiophanate-methyl,Azoxystrobin, Carpropamid, Edifenphos, Ferimzone, Iprobenfos,Isoprothiolane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole,Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tiadinil; CottonHerbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn,Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate,Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron,Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; CottonInsecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin,Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid,Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin,Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl,Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin,Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran,Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen,Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides:Etridiazole, Metalaxyl, Quintozene; Soybean Herbicides: Alachlor,Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl,Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin,Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim,Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl,Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole,Deltamethrin, β-Cyfluthrin, gamma and lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb,beta-Cyfluthrin; Soybean Funqicides: Azoxystrobin, Cyproconazole,Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole,Trifloxystrobin, Prothioconazole, Tetraconazole; Sugarbeet Herbicides:Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate,Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim,Triflusulfuron, Tepraloxydim, Quizalofop; Sugarbeet Insecticides:Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid,Dinetofuran, Deltamethrin, β-Cyfluthrin, gamma/lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran; CanolaHerbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate,Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop,Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Carbendazim,Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Canola Insecticides:Carbofuran organophosphates, Pyrethroids, Thiacloprid, Deltamethrin,Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran,β-Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole,Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.

In some embodiments the herbicide is Atrazine, Bromacil, Diuron,Chlorsulfuron, Metsulfuron, Thifensulfuron Methyl, Tribenuron,Acetochlor, Dicamba, Isoxaflutole, Nicosulfuron, Rimsulfuron,Pyrithiobac-sodium, Flumioxazin, Chlorimuron-Ethyl, Metribuzin,Quizalofop, S-metolachlor, Hexazinne or combinations thereof.

In some embodiments the insecticide is Esfenvalerate,Chlorantraniliprole, Methomyl, Indoxacarb, Oxamyl or combinationsthereof.

Pesticidal and Insecticidal Activity

“Pest” includes but is not limited to, insects, fungi, bacteria,nematodes, mites, ticks and the like. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyLepidoptera and Coleoptera.

Those skilled in the art will recognize that not all compounds areequally effective against all pests. Compounds of the embodimentsdisplay activity against insect pests, which may include economicallyimportant agronomic, forest, greenhouse, nursery ornamentals, food andfiber, public and animal health, domestic and commercial structure,household and stored product pests.

Larvae of the order Lepidoptera include, but are not limited to,armyworms, cutworms, loopers and heliothines in the family NoctuidaeSpodoptera frugiperda JE Smith (fall armyworm); S. exigua Hübner (beetarmyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar);Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus(cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogoniaMorrison (western cutworm); A. subterranea Fabricius (granulatecutworm); Alabama argillacea Hübner (cotton leaf worm); Trichoplusia niHübner (cabbage looper); Pseudoplusia includens Walker (soybean looper);Anticarsia gemmatalis Hübner (velvetbean caterpillar); Hypena scabraFabricius (green cloverworm); Heliothis virescens Fabricius (tobaccobudworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindaraBarnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris(darksided cutworm); Earias insulana Boisduval (spiny bollworm); E.vittella Fabricius (spotted bollworm); Helicoverpa armigera Hübner(American bollworm); H. zea Boddie (corn earworm or cotton bollworm);Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialisGrote (citrus cutworm); borers, casebearers, webworms, coneworms, andskeletonizers from the family Pyralidae Ostrinia nubilalis Hübner(European corn borer); Amyelois transitella Walker (naval orangeworm);Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautellaWalker (almond moth); Chilo suppressalis Walker (rice stem borer); C.partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth);Crambus caliginosellus Clemens (corn root webworm); C. teterrellusZincken (bluegrass webworm); Cnaphalocrocis medinalis Guenée (rice leafroller); Desmia funeralis Hübner (grape leaffolder); Diaphania hyalinataLinnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraeagrandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius(surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestiaelutella Hübner (tobacco (cacao) moth); Galleria mellonella Linnaeus(greater wax moth); Herpetogramma licarsisalis Walker (sod webworm);Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellusZeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser waxmoth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalisWalker (tea tree web moth); Maruca testulalis Geyer (bean pod borer);Plodia interpunctella Hübner (Indian meal moth); Scirpophaga incertulasWalker (yellow stem borer); Udea rubigalis Guenée (celery leaftier); andleafrollers, budworms, seed worms and fruit worms in the familyTortricidae Acleris gloverana Walsingham (Western blackheaded budworm);A. variana Fernald (Eastern blackheaded budworm); Archips argyrospilaWalker (fruit tree leaf roller); A. rosana Linnaeus (European leafroller); and other Archips species, Adoxophyes orana Fischer vonRösslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham(banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C.pomonella Linnaeus (coding moth); Platynota flavedana Clemens(variegated leafroller); P. stultana Walsingham (omnivorous leafroller);Lobesia botrana Denis & Schiffermüller (European grape vine moth);Spilonota ocellana Denis & Schiffermüller (eyespotted bud moth);Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguellaHübner (vine moth); Bonagota salubricola Meyrick (Brazilian appleleafroller); Grapholita molesta Busck (oriental fruit moth); Suleimahelianthana Riley (sunflower bud moth); Argyrotaenia spp.; Choristoneuraspp.

Selected other agronomic pests in the order Lepidoptera include, but arenot limited to, Alsophila pometaria Harris (fall cankerworm); Anarsialineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith(orange striped oakworm); Antheraea pernyi Guérin-Méneville (Chinese OakTussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiellaBusck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfacaterpillar); Datana integerrima Grote & Robinson (walnut caterpillar);Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomossubsignaria Hübner (elm spanworm); Erannis tiliaria Harris (lindenlooper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisinaamericana Guérin-Méneville (grapeleaf skeletonizer); Hemileuca oliviaeCockrell (range caterpillar); Hyphantria cunea Drury (fall webworm);Keiferia lycopersicella Walsingham (tomato pinworm); Lambdinafiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellarialugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus(satin moth); Lymantria dispar Linnaeus (gypsy moth); Manducaquinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M.sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumataLinnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm);Papilio cresphontes Cramer (giant swallowtail orange dog); Phryganidiacalifornica Packard (California oakworm); Phyllocnistis citrellaStainton (citrus leafminer); Phyllonorycter blancardella Fabricius(spotted tentiform leafminer); Pieris brassicae Linnaeus (large whitebutterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus(green veined white butterfly); Platyptilia carduidactyla Riley(artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth);Pectinophora gossypiella Saunders (pink bollworm); Pontia protodiceBoisduval and Leconte (Southern cabbageworm); Sabulodes aegrotata Guenée(omnivorous looper); Schizura concinna J. E. Smith (red humpedcaterpillar); Sitotroga cerealella Olivier (Angoumois grain moth);Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar);Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick(tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothissubflexa Guenée; Malacosoma spp. and Orgyia spp.

Of interest are larvae and adults of the order Coleoptera includingweevils from the families Anthribidae, Bruchidae and Curculionidae(including, but not limited to: Anthonomus grandis Boheman (bollweevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil);Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (riceweevil); Hypera punctata Fabricius (clover leaf weevil);Cylindrocopturus adspersus LeConte (sunflower stem weevil); Smicronyxfulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (graysunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug));flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetlesand leafminers in the family Chrysomelidae (including, but not limitedto: Leptinotarsa decemlineata Say (Colorado potato beetle); Diabroticavirgifera virgifera LeConte (western corn rootworm); D. barberi Smithand Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber(southern corn rootworm); Chaetocnema pulicaria Melsheimer (corn fleabeetle); Phyllotreta cruciferae Goeze (Crucifer flea beetle);Phyllotreta striolata (stripped flea beetle); Colaspis brunnea Fabricius(grape colaspis); Oulema melanopus Linnaeus (cereal leaf beetle);Zygogramma exclamationis Fabricius (sunflower beetle)); beetles from thefamily Coccinellidae (including, but not limited to: Epilachnavarivestis Mulsant (Mexican bean beetle)); chafers and other beetlesfrom the family Scarabaeidae (including, but not limited to: Popilliajaponica Newman (Japanese beetle); Cyclocephala borealis Arrow (northernmasked chafer, white grub); C. immaculata Olivier (southern maskedchafer, white grub); Rhizotrogus majalis Razoumowsky (European chafer);Phyllophaga crinita Burmeister (white grub); Ligyrus gibbosus De Geer(carrot beetle)); carpet beetles from the family Dermestidae; wirewormsfrom the family Elateridae, Eleodes spp., Melanotus spp.; Conoderusspp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; barkbeetles from the family Scolytidae and beetles from the familyTenebrionidae.

Adults and immatures of the order Diptera are of interest, includingleafminers Agromyza parvicornis Loew (corn blotch leafminer); midges(including, but not limited to: Contarinia sorghicola Coquillett(sorghum midge); Mayetiola destructor Say (Hessian fly); Sitodiplosismosellana Géhin (wheat midge); Neolasioptera murtfeldtiana Felt,(sunflower seed midge)); fruit flies (Tephritidae), Oscinella fritLinnaeus (fruit flies); maggots (including, but not limited to: Deliaplatura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly)and other Delia spp., Meromyza americana Fitch (wheat stem maggot);Musca domestica Linnaeus (house flies); Fannia canicularis Linnaeus, F.femoralis Stein (lesser house flies); Stomoxys calcitrans Linnaeus(stable flies)); face flies, horn flies, blow flies, Chrysomya spp.;Phormia spp. and other muscoid fly pests, horse flies Tabanus spp.; botflies Gastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma spp.; deerflies Chrysops spp.; Melophagus ovinus Linnaeus (keds) and otherBrachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; blackflies Prosimulium spp.; Simulium spp.; biting midges, sand flies,sciarids, and other Nematocera.

Included as insects of interest are adults and nymphs of the ordersHemiptera and Homoptera such as, but not limited to, adelgids from thefamily Adelgidae, plant bugs from the family Miridae, cicadas from thefamily Cicadidae, leafhoppers, Empoasca spp.; from the familyCicadellidae, planthoppers from the families Cixiidae, Flatidae,Fulgoroidea, Issidae and Delphacidae, treehoppers from the familyMembracidae, psyllids from the family Psyllidae, whiteflies from thefamily Aleyrodidae, aphids from the family Aphididae, phylloxera fromthe family Phylloxeridae, mealybugs from the family Pseudococcidae,scales from the families Asterolecanidae, Coccidae, Dactylopiidae,Diaspididae, Eriococcidae Ortheziidae, Phoenicococcidae andMargarodidae, lace bugs from the family Tingidae, stink bugs from thefamily Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs fromthe family Lygaeidae, spittlebugs from the family Cercopidae squash bugsfrom the family Coreidae and red bugs and cotton stainers from thefamily Pyrrhocoridae.

Agronomically important members from the order Homoptera furtherinclude, but are not limited to: Acyrthisiphon pisum Harris (pea aphid);Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black beanaphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicisForbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecolaPatch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid);Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxiaKurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantagineaPaaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly appleaphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopteruspruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnipaphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphumeuphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach-potatoaphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid);Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch(corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphisgraminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcaneaphid); Sitobion avenae Fabricius (English grain aphid); Therioaphismaculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer deFonscolombe (black citrus aphid) and T. citricida Kirkaldy (brown citrusaphid); Adelges spp. (adelgids); Phylloxera devastatrix Pergande (pecanphylloxera); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotatowhitefly); B. argentifolii Bellows & Perring (silverleaf whitefly);Dialeurodes citri Ashmead (citrus whitefly); Trialeurodes abutiloneus(bandedwinged whitefly) and T. vaporariorum Westwood (greenhousewhitefly); Empoasca fabae Harris (potato leafhopper); Laodelphaxstriatellus Fallen (smaller brown planthopper); Macrolestesquadrilineatus Forbes (aster leafhopper); Nephotettix cinticeps Uhler(green leafhopper); N. nigropictus Stål (rice leafhopper); Nilaparvatalugens Stål (brown planthopper); Peregrinus maidis Ashmead (cornplanthopper); Sogatella furcifera Horvath (white-backed planthopper);Sogatodes orizicola Muir (rice delphacid); Typhlocyba pomaria McAtee(white apple leafhopper); Erythroneoura spp. (grape leafhoppers);Magicicada septendecim Linnaeus (periodical cicada); Icerya purchasiMaskell (cottony cushion scale); Quadraspidiotus perniciosus Comstock(San Jose scale); Planococcus citri Risso (citrus mealybug);Pseudococcus spp. (other mealybug complex); Cacopsylla pyricola Foerster(pear psylla); Trioza diospyri Ashmead (persimmon psylla).

Agronomically important species of interest from the order Hemipterainclude, but are not limited to: Acrosternum hilare Say (green stinkbug); Anasa tristis De Geer (squash bug); Blissus leucopterusleucopterus Say (chinch bug); Corythuca gossypii Fabricius (cotton lacebug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellusHerrich-Schiffer (cotton stainer); Euschistus servus Say (brown stinkbug); E. variolarius Palisot de Beauvois (one-spotted stink bug);Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say(leaf-footed pine seed bug); Lygus lineolaris Palisot de Beauvois(tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug);L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius(European tarnished plant bug); Lygocoris pabulinus Linnaeus (commongreen capsid); Nezara viridula Linnaeus (southern green stink bug);Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas(large milkweed bug); Pseudatomoscelis seriatus Reuter (cottonfleahopper).

Furthermore, embodiments may be effective against Hemiptera such,Calocoris norvegicus Gmelin (strawberry bug); Orthops campestrisLinnaeus; Plesiocoris rugicollis Fallen (apple capsid); Cyrtopeltismodestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly);Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Diaphnocorischlorionis Say (honeylocust plant bug); Labopidicola allii Knight (onionplant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper);Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatusFabricius (four-lined plant bug); Nysius ericae Schilling (false chinchbug); Nysius raphanus Howard (false chinch bug); Nezara viridulaLinnaeus (Southern green stink bug); Eurygaster spp.; Coreidae spp.;Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp.and Cimicidae spp.

Also included are adults and larvae of the order Acari (mites) such asAceria tosichella Keifer (wheat curl mite); Petrobia latens Müller(brown wheat mite); spider mites and red mites in the familyTetranychidae, Panonychus ulmi Koch (European red mite); Tetranychusurticae Koch (two spotted spider mite); (T. mcdanieli McGregor (McDanielmite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestaniUgarov & Nikolski (strawberry spider mite); flat mites in the familyTenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust andbud mites in the family Eriophyidae and other foliar feeding mites andmites important in human and animal health, i.e., dust mites in thefamily Epidermoptidae, follicle mites in the family Demodicidae, grainmites in the family Glycyphagidae, ticks in the order Ixodidae. Ixodesscapularis Say (deer tick); I. holocyclus Neumann (Australian paralysistick); Dermacentor variabilis Say (American dog tick); Amblyommaamericanum Linnaeus (lone star tick) and scab and itch mites in thefamilies Psoroptidae, Pyemotidae and Sarcoptidae.

Insect pests of the order Thysanura are of interest, such as Lepismasaccharina Linnaeus (silverfish); Thermobia domestica Packard(firebrat).

Additional arthropod pests covered include: spiders in the order Araneaesuch as Loxosceles reclusa Gertsch and Mulaik (brown recluse spider) andthe Latrodectus mactans Fabricius (black widow spider) and centipedes inthe order Scutigeromorpha such as Scutigera coleoptrata Linnaeus (housecentipede).

Insect pest of interest include the superfamily of stink bugs and otherrelated insects including but not limited to species belonging to thefamily Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorusguildini, Euschistus servus, Acrosternum hilare, Euschistus heros,Euschistus tristigmus, Acrosternum hilare, Dichelops furcatus, Dichelopsmelacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae(Megacopta cribraria—Bean plataspid) and the family Cydnidae(Scaptocoris castanea—Root stink bug) and Lepidoptera species includingbut not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie;soybean looper, e.g., Pseudoplusia includens Walker and velvet beancaterpillar e.g., Anticarsia gemmatalis Hübner.

Methods for measuring pesticidal activity are well known in the art.See, for example, Czapla and Lang, (1990) J. Econ. Entomol.83:2480-2485; Andrews, et al., (1988) Biochem. J. 252:199-206; Marrone,et al., (1985) J. of Economic Entomology 78:290-293 and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety. Generally, the protein is mixed and used in feeding assays.See, for example Marrone, et al., (1985) J. of Economic Entomology78:290-293. Such assays can include contacting plants with one or morepests and determining the plant's ability to survive and/or cause thedeath of the pests.

Nematodes include parasitic nematodes such as root-knot, cyst and lesionnematodes, including Heterodera spp., Meloidogyne spp. and Globoderaspp.; particularly members of the cyst nematodes, including, but notlimited to, Heterodera glycines (soybean cyst nematode); Heteroderaschachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode)and Globodera rostochiensis and Globodera pailida (potato cystnematodes). Lesion nematodes include Pratylenchus spp.

Seed Treatment

To protect and to enhance yield production and trait technologies, seedtreatment options can provide additional crop plan flexibility and costeffective control against insects, weeds and diseases. Seed material canbe treated, typically surface treated, with a composition comprisingcombinations of chemical or biological herbicides, herbicide safeners,insecticides, fungicides, germination inhibitors and enhancers,nutrients, plant growth regulators and activators, bactericides,nematocides, avicides and/or molluscicides. These compounds aretypically formulated together with further carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation. The coatings may be applied by impregnating propagationmaterial with a liquid formulation or by coating with a combined wet ordry formulation. Examples of the various types of compounds that may beused as seed treatments are provided in The Pesticide Manual: A WorldCompendium, C. D. S. Tomlin Ed., Published by the British CropProduction Council, which is hereby incorporated by reference.

Some seed treatments that may be used on crop seed include, but are notlimited to, one or more of abscisic acid, acibenzolar-S-methyl,avermectin, amitrol, azaconazole, azospirillum, azadirachtin,azoxystrobin, Bacillus spp. (including one or more of cereus, firmus,megaterium, pumilis, sphaericus, subtilis and/or thuringiensis species),bradyrhizobium spp. (including one or more of betae, canariense,elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/oryuanmingense), captan, carboxin, chitosan, clothianidin, copper,cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil,fluoxastrobin, fluquinconazole, flurazole, fluxofenim, harpin protein,imazalil, imidacloprid, ipconazole, isoflavenoids,lipo-chitooligosaccharide, mancozeb, manganese, maneb, mefenoxam,metalaxyl, metconazole, myclobutanil, PCNB, penflufen, penicillium,penthiopyrad, permethrine, picoxystrobin, prothioconazole,pyraclostrobin, rynaxypyr, S-metolachlor, saponin, sedaxane, TCMTB,tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram,tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin,triticonazole and/or zinc. PCNB seed coat refers to EPA RegistrationNumber 00293500419, containing quintozen and terrazole. TCMTB refers to2-(thiocyanomethylthio) benzothiazole.

Seed varieties and seeds with specific transgenic traits may be testedto determine which seed treatment options and application rates maycomplement such varieties and transgenic traits in order to enhanceyield. For example, a variety with good yield potential but head smutsusceptibility may benefit from the use of a seed treatment thatprovides protection against head smut, a variety with good yieldpotential but cyst nematode susceptibility may benefit from the use of aseed treatment that provides protection against cyst nematode, and soon. Likewise, a variety encompassing a transgenic trait conferringinsect resistance may benefit from the second mode of action conferredby the seed treatment, a variety encompassing a transgenic traitconferring herbicide resistance may benefit from a seed treatment with asafener that enhances the plants resistance to that herbicide, etc.Further, the good root establishment and early emergence that resultsfrom the proper use of a seed treatment may result in more efficientnitrogen use, a better ability to withstand drought and an overallincrease in yield potential of a variety or varieties containing acertain trait when combined with a seed treatment.

Methods for Killing an Insect Pest and Controlling an Insect Population

In some embodiments methods are provided for killing an insect pest,comprising contacting the insect pest, either simultaneously orsequentially, with an insecticidally-effective amount of a recombinantIPD090 polypeptide or IPD090 chimeric polypeptide of the disclosure. Insome embodiments methods are provided for killing an insect pest,comprising contacting the insect pest with an insecticidally-effectiveamount of a recombinant pesticidal protein of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 379, SEQ ID NO: 384 or a variant thereof.

In some embodiments methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population, eithersimultaneously or sequentially, with an insecticidally-effective amountof a recombinant IPD090 polypeptide or IPD090 chimeric polypeptide ofthe disclosure. In some embodiments methods are provided for controllingan insect pest population, comprising contacting the insect pestpopulation with an insecticidally-effective amount of a recombinantIPD090 polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ, SEQID NO: 379, SEQ ID NO: 384 or a variant thereof. As used herein,“controlling a pest population” or “controls a pest” refers to anyeffect on a pest that results in limiting the damage that the pestcauses. Controlling a pest includes, but is not limited to, killing thepest, inhibiting development of the pest, altering fertility or growthof the pest in such a manner that the pest provides less damage to theplant, decreasing the number of offspring produced, producing less fitpests, producing pests more susceptible to predator attack or deterringthe pests from eating the plant.

In some embodiments methods are provided for controlling an insect pestpopulation resistant to a pesticidal protein, comprising contacting theinsect pest population, either simultaneously or sequentially, with aninsecticidally-effective amount of a recombinant IPD090 polypeptide orchimeric IPD090 polypeptide of the disclosure. In some embodimentsmethods are provided for controlling an insect pest population resistantto a pesticidal protein, comprising contacting the insect pestpopulation with an insecticidally-effective amount of a recombinantIPD090 polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 379, SEQ ID NO: 384 or a variant thereof.

In some embodiments methods are provided for protecting a plant from aninsect pest, comprising expressing in the plant or cell thereof at leastone recombinant polynucleotide encoding an IPD090 polypeptide orchimeric IPD090 polypeptide. In some embodiments methods are providedfor protecting a plant from an insect pest, comprising expressing in theplant or cell thereof a recombinant polynucleotide encoding IPD090polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379,SEQ ID NO: 384 or variants thereof.

In some embodiments methods are provided for protecting a plant from aninsect pest, comprising expressing in the plant or cell thereof arecombinant polynucleotide encoding the polypeptide of SEQ ID NO: 385,SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 388 or variants thereof. Insome embodiments methods are provided for protecting a plant from aninsect pest, comprising expressing in the plant or cell thereof therecombinant polynucleotide of SEQ ID NO: 381, SEQ ID NO: 382 or SEQ IDNO: 383.

Insect Resistance Management (IRM) Strategies Expression of B.thuringiensis δ-endotoxins in transgenic corn plants has proven to be aneffective means of controlling agriculturally important insect pests(Perlak, et al., 1990; 1993). However, insects have evolved that areresistant to B. thuringiensis δ-endotoxins expressed in transgenicplants. Such resistance, should it become widespread, would clearlylimit the commercial value of germplasm containing genes encoding suchB. thuringiensis 6-endotoxins.

One way to increasing the effectiveness of the transgenic insecticidesagainst target pests and contemporaneously reducing the development ofinsecticide-resistant pests is to use provide non-transgenic (i.e.,non-insecticidal protein) refuges (a section of non-insecticidalcrops/corn) for use with transgenic crops producing a singleinsecticidal protein active against target pests. The United StatesEnvironmental Protection Agency(epa.gov/oppbppdl/biopesticides/pips/bt_corn_refuge_2006.htm, which canbe accessed using the www prefix) publishes the requirements for usewith transgenic crops producing a single Bt protein active againsttarget pests. In addition, the National Corn Growers Association, ontheir website:(ncga.com/insect-resistance-management-fact-sheet-bt-corn, which can beaccessed using the www prefix) also provides similar guidance regardingrefuge requirements. Due to losses to insects within the refuge area,larger refuges may reduce overall yield.

Another way of increasing the effectiveness of the transgenicinsecticides against target pests and contemporaneously reducing thedevelopment of insecticide-resistant pests would be to have a repositoryof insecticidal genes that are effective against groups of insect pestsand which manifest their effects through different modes of action.

Expression in a plant of two or more insecticidal compositions toxic tothe same insect species, each insecticide being expressed at efficaciouslevels would be another way to achieve control of the development ofresistance. This is based on the principle that evolution of resistanceagainst two separate modes of action is far more unlikely than only one.Roush, for example, outlines two-toxin strategies, also called“pyramiding” or “stacking,” for management of insecticidal transgeniccrops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998)353:1777-1786). Stacking or pyramiding of two different proteins eacheffective against the target pests and with little or nocross-resistance can allow for use of a smaller refuge. The USEnvironmental Protection Agency requires significantly less (generally5%) structured refuge of non-Bt corn be planted than for single traitproducts (generally 20%). There are various ways of providing the IRMeffects of a refuge, including various geometric planting patterns inthe fields and in-bag seed mixtures, as discussed further by Roush.

In some embodiments the IPD090 polypeptides of the disclosure are usefulas an insect resistance management strategy in combination (i.e.,pyramided) with other pesticidal proteins include but are not limited toBt toxins, Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins,other insecticidally active proteins, and the like.

Provided are methods of controlling Lepidoptera and/or Coleoptera insectinfestation(s) in a transgenic plant that promote insect resistancemanagement, comprising expressing in the plant at least two differentinsecticidal proteins having different modes of action.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management comprises the presentation of at least one of theIPD090 polypeptide insecticidal proteins to insects in the orderLepidoptera and/or Coleoptera.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management comprises the presentation of at least one of theIPD090 polypeptides of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 379, SEQ ID NO: 384 or variants thereof, insecticidal to insects inthe order Lepidoptera and/or Coleoptera.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management comprise expressing in the transgenic plant anIPD090 polypeptide and a Cry protein or other insecticidal protein toinsects in the order Lepidoptera and/or Coleoptera having differentmodes of action.

In some embodiments the methods, of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management, comprise expression in the transgenic plant anIPD090 polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 379, SEQ ID NO: 384 or variants thereof and a Cry protein or otherinsecticidal protein to insects in the order Lepidoptera and/orColeoptera, where the IPD090 polypeptide and Cry protein have differentmodes of action.

Also provided are methods of reducing likelihood of emergence ofLepidoptera and/or Coleoptera insect resistance to transgenic plantsexpressing in the plants insecticidal proteins to control the insectspecies, comprising expression of an IPD090 polypeptide insecticidal tothe insect species in combination with a second insecticidal protein tothe insect species having different modes of action.

Also provided are means for effective Lepidoptera and/or Coleopterainsect resistance management of transgenic plants, comprisingco-expressing at high levels in the plants two or more insecticidalproteins toxic to Lepidoptera and/or Coleoptera insects but eachexhibiting a different mode of effectuating its killing activity,wherein the two or more insecticidal proteins comprise an IPD090polypeptide and a Cry protein. Also provided are means for effectiveLepidoptera and/or Coleoptera insect resistance management of transgenicplants, comprising co-expressing at high levels in the plants two ormore insecticidal proteins toxic to Lepidoptera and/or Coleopterainsects but each exhibiting a different mode of effectuating its killingactivity, wherein the two or more insecticidal proteins comprise anIPD090 polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 379, SEQ ID NO: 384 or variants thereof and a Cry protein or otherinsecticidally active protein.

In addition, methods are provided for obtaining regulatory approval forplanting or commercialization of plants expressing proteins insecticidalto insects in the order Lepidoptera and/or Coleoptera, comprising thestep of referring to, submitting or relying on insect assay binding datashowing that the IPD090 polypeptide does not compete with binding sitesfor Cry proteins in such insects. In addition, methods are provided forobtaining regulatory approval for planting or commercialization ofplants expressing proteins insecticidal to insects in the orderLepidoptera and/or Coleoptera, comprising the step of referring to,submitting or relying on insect assay binding data showing that theIPD090 polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 379, SEQ ID NO: 384 or variant thereof does not compete with bindingsites for Cry proteins in such insects.

Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods compriseproviding a plant or plant cell expressing a polynucleotide encoding thepesticidal polypeptide sequence disclosed herein and growing the plantor a seed thereof in a field infested with a pest against which thepolypeptide has pesticidal activity. In some embodiments, thepolypeptide has pesticidal activity against a Lepidopteran, Coleopteran,Dipteran, Hemipteran or nematode pest, and the field is infested with aLepidopteran, Hemipteran, Coleopteran, Dipteran or nematode pest.

As defined herein, the “yield” of the plant refers to the quality and/orquantity of biomass produced by the plant. “Biomass” as used hereinrefers to any measured plant product. An increase in biomass productionis any improvement in the yield of the measured plant product.Increasing plant yield has several commercial applications. For example,increasing plant leaf biomass may increase the yield of leafy vegetablesfor human or animal consumption. Additionally, increasing leaf biomasscan be used to increase production of plant-derived pharmaceutical orindustrial products. An increase in yield can comprise any statisticallysignificant increase including, but not limited to, at least a 1%increase, at least a 3% increase, at least a 5% increase, at least a 10%increase, at least a 20% increase, at least a 30%, at least a 50%, atleast a 70%, at least a 100% or a greater increase in yield compared toa plant not expressing the pesticidal sequence.

In specific methods, plant yield is increased as a result of improvedpest resistance of a plant expressing an IPD090 polypeptide disclosedherein. Expression of the IPD090 polypeptide results in a reducedability of a pest to infest or feed on the plant, thus improving plantyield.

Methods of Processing

Further provided are methods of processing a plant, plant part or seedto obtain a food or feed product from a plant, plant part or seedcomprising an IPD090 polynucleotide. The plants, plant parts or seedsprovided herein, can be processed to yield oil, protein products and/orby-products that are derivatives obtained by processing that havecommercial value. Non-limiting examples include transgenic seedscomprising a nucleic acid molecule encoding an IPD090 polypeptide whichcan be processed to yield soy oil, soy products and/or soy by-products.

“Processing” refers to any physical and chemical methods used to obtainany soy product and includes, but is not limited to, heat conditioning,flaking and grinding, extrusion, solvent extraction or aqueous soakingand extraction of whole or partial seeds

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTALS Example 1—Identification of an Insecticidal Protein ActiveAgainst Western Corn Rootworm (Diabrotica virqifera virgiferaLeConte—WCRW) from Strain JH34071-1

The insecticidal protein designated as IPD090Aa was identified byprotein purification, N-terminal amino acid sequencing, and PCR cloningfrom Pseudomonas sp. strain JH34071-1 as follows. Insecticidal activityagainst WCRW was observed from a cell lysate of JH34071-1 that was grownin Tryptic Soy broth (TSB, peptone from casein 15 g/L; peptone fromsoymeal 5.0 g/L; NaCl 5.0 g/L) and cultured 1 day at 28° C. with shakingat 200 rpm. This insecticidal activity exhibited heat and proteasesensitivity indicating a proteinaceous nature.

Bioassays with WCRW were conducted using the cell lysate samples mixedwith Diabrotica diet (Frontier Agricultural Sciences, Newark, Del.) in a96 well format. WCRW neonates were placed into each well of a 96 wellplate. The assay was run four days at 25° C. and then was scored forinsect mortality and stunting of insect growth. The scores were noted asdead (3), severely stunted (2) (little or no growth but alive), stunted(1) (growth to second instar but not equivalent to controls) or noactivity (0). Samples demonstrating mortality or severe stunting werefurther studied.

Genomic DNA of isolated strain JH34071-1 was prepared according to alibrary construction protocol developed by Illumina and sequenced usingthe Illumina® Genome Analyzer IIx (Illumina Inc., 9885 Towne CenterDrive, San Diego, Calif. 92121). The nucleic acid contig sequences wereassembled and open reading frames were generated. The 16S ribosomal DNAsequence of strain JH34071-1 was BLAST™ searched against the NCBIdatabase identifying strain JH34071-1 as a Pseudomonas species.

Cell pellets of strain JH34071-1 were homogenized at 30,000 psi afterre-suspension in 20 mM Tris buffer, pH 8 with “Complete, EDTA-free”protease inhibitor cocktail (Roche, Indianapolis, Ind.). The crudelysate was cleared by centrifugation and brought to 75% saturation withammonium sulfate. The 75% ammonium sulfate solution was then centrifugedand the supernatant was discarded. The pellet portion was suspended in20 mM Tris pH 8.0 and then brought to 1.5 M ammonium sulfate with theaddition of a 2 M ammonium sulfate, 20 mM Tris pH 8.0 solution. Thissolution was clarified and loaded onto a TSKgel™ Phenyl-5PW column(Tosoh Bioscience, Tokyo, Japan) equilibrated in 20 mM Tris pH 8.0, 1.5M ammonium sulfate. Insecticidal activity eluted with a gradient to 20mM Tris, pH 8. Active fractions were pooled, concentrated on 10 kDamolecular weight cutoff centrifugal concentrators (Sartorius Stedim,Goettingen, Germany) and desalted into 20 mM piperazine pH 9.5 using aSephadex G25 (GE Healthcare, Piscataway, N.J.) column. The desalted poolwas loaded onto a Mono Q™ column (GE Healthcare, Piscataway, N.J.)equilibrated in 20 mM piperazine, pH 9.5 and eluted with a gradient of 0to 0.4 M NaCl. Active fractions were pooled and loaded onto a Superdex™200 column (GE Healthcare) equilibrated in phosphate buffered saline(PBS). SDS-PAGE analysis of fractions indicated that WCRW activitycoincided with a prominent band after staining with GelCode™ Blue StainReagent (Thermo Fisher Scientific®). The protein band was excised,digested with trypsin and analyzed by nano-liquidchromatography/electrospray tandem mass spectrometry (nano-LC/ESI-MS/MS)on a Thermo Q Exactive™ Orbitrap™ mass spectrometer (Thermo FisherScientific®, 81 Wyman Street, Waltham, Mass. 02454) interfaced with anEksigent™ NanoLC™ 1-D Plus nano-Ic system (AB Sciex™, 500 OldConnecticut Path, Framingham, Mass. 01701). Protein identification wasdone by internal database searches using Mascot® (Matrix Science, 10Perrins Lane, London NW3 1QY UK), which identified the IPD090Aapolypeptide (SEQ ID NO: 2) encoded by the polynucleotide of SEQ IDNO: 1. Cloning and recombinant expression confirmed the insecticidalactivity of the IPD090Aa polypeptide (SEQ ID NO: 2) against WCRW.

Example 2—Identification of Homologs of IPD090Aa

Gene identities may be determined by conducting BLAST™ (Basic LocalAlignment 20 Search Tool; Altschul, et al. (1993) J. Molec. Biol. 215:403-410; see also ncbi.nlm.nih.gov/BLAST/, which can be accessed usingthe www prefix) searches under default parameters for similarity tosequences contained in the publically available BLAST database(comprising all non-redundant GenBank CDS translations, sequencesderived from the 3-Dimensional Brookhaven Data Bank, and DDBJ databases.In addition to public databases DuPont Pioneer databases were searched.IPD090Aa (SEQ ID NO: 2) showed distant homology to proteins which have aPfam ID #PF01823 which have membrane attack complex/perforin (MACPF)domains (Reference to Pfam database: en.wikipedia.org/wiki/Pfam, whichcan be accessed using the www prefix). Several homologs of the IPD090Aapolypeptide (SEQ ID NO: 2) identified having varying percent identityare shown in Table 1. Table 2 shows a matrix table of pair-wise identityrelationships for global alignments of the IPD090 homologs, based uponthe ClustalW algorithm implemented using the in the ALIGNX® module ofthe Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, Calif.)with all default parameters.

TABLE 1 Protein Identity to identifier IPD090Aa Strain identifierSpecies Polynucleotide Polypeptide IPD090Aa JH34071-1, SSP342A9-1Pseudomonas sp. SEQ ID NO: 1 SEQ ID NO: 2 IPD090Ab 99.8% SS342A7-1Pseudomonas SEQ ID NO: 3 SEQ ID NO: 4 monteilii IPD090Ca 79.3%JH23589-1, JH23611-2, Pseudomonas SEQ ID NO: 5 SEQ ID NO: 6 JH23959-1,JH59556-2, entomophila JH61488-2, JH62159-2, JH62167-1, JH62246-2,JH62258-1, JH62270-2, and JH62417-2 IPD090Fa 49.4% GenBank Accession #Woodsholea SEQ ID NO: 7 SEQ ID NO: 8 WP_019961352 maritima IPD090Ac89.1% SSP1049E7- Pseudomonas SEQ ID NO: 380 SEQ ID NO: 384 monteiliiIPD090Ga 38.9 GenBank Accession # Clavibacter SEQ ID NO: 381 SEQ ID NO:385 WP_012039071 michiganensis IPD090Gb 35.6 GenBank Accession #Serratia SEQ ID NO: 382 SEQ ID NO: 386 WP_012145116 proteamaculansIPD090Gc 37.9 GenBank Accession # Marinomonas sp. SEQ ID NO: 383 SEQ IDNO: 387 WP_046018755.1 IPD090Gd 36.2 GenBank Accession # Serratia SEQ IDNO: 388 WP_073439185 plymuthica

TABLE 2 IPD090Ab IPD090Ac IPD090Ca IPD090Cd IPD090Fa IPD090Ga IPD090GbIPD090Gc IPD090Gd SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQID SEQ ID NO: 4 NO: NO: 6 NO: 379 NO: 8 NO: 385 NO: 386 NO: 387 NO: 388IPD090Aa SEQ ID NO: 2 99.8 89.1 79.3 79.8 50.8 38.9 35.6 37.9 36.2IPD090Ab SEQ ID NO: 4 — 88.9 79.1 79.5 50.8 38.7 35.4 37.7 36.0 IPD090AcSEQ ID NO: 384 — — 76.8 77.6 48.9 36.8 35.1 36.6 35.4 IPD090Ca SEQ IDNO: 6 — — — 80.3 48.5 36.2 35.5 36.9 36.9 IPD090Cd SEQ ID NO: 379 — — —— 47.2 37.7 36.3 37.7 38.7 IPD090Fa SEQ ID NO: 8 — — — — — 34.5 34.339.4 35.3 IPD090Ga SEQ ID NO: 385 — — — — — — 34.3 32.7 33.5 IPD090GbSEQ ID NO: 386 — — — — — — — 33.5 82.8 IPD090Gc SEQ ID NO: 387 — — — — —— — — 32.9

Genome sequencing of a pool of bacterial strains identified thepolynucleotide of SEQ ID NO: 378 encoding the IPD090 homolog, IPD090Cd(SEQ ID NO: 379) having ˜80% amino acid sequence identity to IPD090Ca(SEQ ID NO: 6).

Example 3—E. coli Expression of IPD090Aa

Peptide fragments from MS analysis were used to locate the IPD090Aacoding sequence (SEQ ID NO: 1) within the JH34071-1 contig.Additionally, N-terminal sequencing was used to confirm the predictedstart site. The coding sequence was used to design the primers CTB54-FOR(SEQ ID NO: 354) and CTB55-REV (SEQ ID NO: 355) to clone the IPD090Aacoding sequence (SEQ ID NO: 1) (with the native stop codon TAG) intopET-24a (Novagen®) for untagged translation and pET-14b (Novagen®) foran N-terminal translation of a 6×-His tag using NdeI/XhoI sites.Additionally, the coding sequence was used to design the primersCTB54-FOR (SEQ ID NO: 354) and CTB56-REV (SEQ ID NO: 356) to clone theIPD090Aa coding sequence (SEQ ID NO: 1) (with no stop codon) intopET-24a (Novagen®) for a C-terminal translation of a 6×-His tag usingNdeI/XhoI sites. The KOD Hot Start Master Mix (EMD Biosciences, SanDiego, Calif.) was used for PCR amplification of the IPD090Aa gene on aBio-Rad™ C1000 Touch™ thermal cycler. Cycling parameters are as follows:1 cycle at 95° C. for 2 minutes; 35 cycles of 95° C. for 20 seconds, 60°C. for 10 seconds and 70° C. for 15 seconds; 1 cycle at 70° C. for 2minutes. Amplicons were gel purified, ligated (T4 DNA Ligase, NewEngland BioLabs, Ipswich, Mass.) into expression vectors (as describedabove), transformed into E. coli One Shot® TOP10 high efficiencychemically competent cells (Invitrogen™-Thermo Fisher Scientific, 81Wyman Street, Waltham, Mass.) and clones were confirmed by sequencing.

The IPD090Aa N-terminal 6× His (SEQ ID NO: 346) and IPD090Aa C-terminal6×-His (SEQ ID NO: 348) expressing constructs were transformed into E.coli BL21 (DE3, Agilent, Santa Clara Calif.) expression cells. One LiterLuria Broth cultures (containing the appropriate antibiotic) were grownuntil an OD₆₀₀ of approximately 0.6 was reached and then the cultureswere induced with 0.3 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG)and allowed to grow for an additional 18 hours at 16° C., 100 rpm. Thecultures were centrifuged at 5,000 rcf for 15 minutes to pellet thecells. Cell pellets were lysed with ¼ B-PER™ II reagent (ThermoScientific), 20 mM Tris pH 8.0, OmniCleave™ endonuclease (Epicentre),ReadyLyse™ lysozyme (Epicentre) and HALT™ Protease Inhibitors (LifeTechnologies) for 30 minutes rocking at room temperature. The lysateswere cleared via centrifugation at 13,000 rcf for 10 minutes and thesupernatants were brought up to 10 mM Imidazole and then applied toseparate 2.5 mL Ni-NTA (QIAGEN® Inc., Valencia, Calif. 91355) columnsequilibrated with PBS, 10 mM imidazole. Columns were washed with 5 mL of20, 40 and 80 mM Imidazole in PBS. Recombinantly expressed IPD090AaN-terminal 6×-His polypeptide (SEQ ID NO: 347) and IPD090Aa C-terminal6×-His polypeptide (SEQ ID NO: 349) were eluted off the columns with 2.5mL of 150 mM imidazole in PBS. Both 2.5 mL eluents were applied toseparate PD10 (GE Healthcare) desalting columns and proteins were elutedoff with 3.5 mL PBS. Purified and desalted IPD090Aa N-terminalpolypeptide (SEQ ID NO: 347) and IPD090Aa C-terminal 6×-His taggedpolypeptide (SEQ ID NO: 349) were submitted to bioassay against WCRW andwere active (Table 3). Additionally, IPD090Aa polypeptide (SEQ ID NO: 2)clear lysate from a 50 mL induction was FPLC-purified and submitted tobioassay against WCRW and was active (see Example 4 below).

Example 4—Purification of Recombinant IPD090Aa Polypeptide

A cell pellet from a 1 L E. coli culture expressing IPD090Aa polypeptide(SEQ ID NO: 2) was suspended in 100 mL 20 mM Tris pH 8.0+1:100 HALT™proteinase inhibitor cocktail (Life Technologies). Cells were lysed at30,000 PSI and the lysate centrifuged at 30,000 g for 30 min. To thesupernatant ammonium sulfate was added to a final concentration of 1.5 Mand the solution allowed to equilibrate overnight. After clarificationthe supernatant was loaded onto a phenyl-5PW column (GE Healthcare,Piscataway, N.J.) equilibrated in 1.5 M ammonium sulfate, 20 mM Tris, pH8.0. The column was washed with 4 column volumes (CV), and IPD090Aapolypeptide (SEQ ID NO: 2) containing fractions eluted with a 10 CVgradient to 20 mM Tris, pH 8.0. Eluate with IPD090Aa polypeptide (SEQ IDNO: 2) was concentrated and further purified by size exclusionchromatography on an S200 column (GE Healthcare, Piscataway, N.J.)equilibrated in PBS. Based on SDS-PAGE, fractions with purified IPD090Aapolypeptide (SEQ ID NO: 2) were combined.

Example 5—Coleoptera Assays with Purified IPD090Aa Protein

Insecticidal activity bioassay screens were conducted with purifiedrecombinant IPD090Aa polypeptide (SEQ ID NO: 2) as well as N-terminallyHis-tagged IPD090Aa polypeptide (SEQ ID NO: 347) and C-terminallyHis-tagged IPD090Aa polypeptide (SEQ ID NO. 349) to evaluate theinsecticidal protein effects on larvae of a variety of Coleopteraincluding Western corn rootworm (Diabrotica virgifera)—WCRW and Northerncorn rootworm (Diabrotica barberi)—NCRW, Coleoptera feeding assays wereconducted on an artificial diet containing the insecticidal protein. Theinsecticidal proteins were incorporated into a Coleopteran-specificartificial diet (Frontier Agricultural Sciences, Newark, Del.). Theproteins were assayed in a dilution series from 188 ppm to 6 ppm. Oneneonate larva was placed in each well to feed ad libitum for 4 days.Each bioassay was done with eight replicates at each dose. Results wereexpressed as positive for larvae reactions such as stunting and ormortality. Results were expressed as negative if the larvae were similarto the negative control that was fed a diet to which the above bufferonly was applied. The average WCRW score for the dilution series from 8assay replicates for the IPD090Aa polypeptide (SEQ ID NO: 2), theIPD090Aa N-terminal 6× His polypeptide (SEQ ID NO: 347), and IPD090AaC-terminal 6×-His polypeptide (SEQ ID NO: 349) are shown in Table 3.

TABLE 3 Polypeptide Avg. Avg. Avg. Purified Concentration WCRW PurifiedPolypeptide WCRW Purified Polypeptide WCRW Polypeptide (ppm) ScorePolypeptide Conc. (ppm) Score Polypeptide Conc. (ppm) Score IPD090Aa 1882.4 IPD090Aa- 188 2.5 IPD090Aa- 188 2.0 SEQ ID NO: 2 131 2.0 N-term 6X131 2.0 C-term 6X 131 2.0 94 2.0 His SEQ ID 94 2.3 His SEQ ID 94 1.8 662.0 NO: 347 66 2.0 NO: 349 66 1.8 47 2.0 47 2.0 47 1.3 33 1.8 33 1.8 331.4 23 1.4 23 2.0 23 1.5 16 1.3 16 1.0 16 1.0 12 0.3 12 1.1 12 0.4 8 0 80.5 8 0.3 6 0 6 0.1 6 0.0 PBS Buffer 0 0 PBS Buffer 0 0 PBS Buffer 0 0Control Control Control

Example 6—E. coli Expression and Insecticidal Activity of anN-Terminally Truncated IPD090Aa Polypeptide

WCRW Bioassays with trypsinized IPD090Aa polypeptide (SEQ ID NO: 2)indicated that a truncated IPD090Aa product was insecticidal. N-terminalsequencing of trypsinized IPD090Aa polypeptide fragment demonstratedthat a polypeptide product starting at alanine 25 of SEQ ID NO: 2 wasformed. A polynucleotide (SEQ ID NO: 9) encoding the IPD090Aa (TR1)polypeptide (SEQ ID NO: 10) was generated by amplifying the IPD090Aagene (SEQ ID NO: 1) using the primers CTB142-FOR (SEQ ID NO: 357) andCTB55-REV (SEQ ID NO: 355) to clone the IPD090Aa (TR1) coding sequence(with the native stop codon TAG) into pET-24a (Novagen) for untaggedtranslation. The KOD Hot Start Master Mix (EMD Biosciences, San Diego,Calif.) was used for PCR amplification of the IPD090Aa (TR1) gene on aBio-Rad® C1000 Touch™ thermal cycler. Cycling parameters are as follows:1 cycle at 95° C. for 2 minutes; 35 cycles of 95° C. for 20 seconds, 60°C. for 10 seconds and 70° C. for 15 seconds; 1 cycle at 70° C. for 2minutes. Amplicons were gel purified, ligated (T4 DNA Ligase, NewEngland BioLabs, Ipswich, Mass.) into expression vectors (as describedabove), transformed into E. coli One Shot® TOP10 high efficiencychemically competent cells (Invitrogen) and clones were confirmed bysequencing.

Confirmed clones expressing the IPD090Aa (TR1) polypeptide (SEQ ID NO:10) were transformed into BL21-Gold expression cells for 1 L inductions.Induction pellets were lysed in 30 mL lysis buffer (20 mM Tris pH 8,¼×B-PER™ II, Omni-Cleave™, Ready-Lyse™ and HALT™ (Life Technologies))rocking at room temp for 1 hour. The lysate was centrifuged at 30,000 gfor 30 min. To the supernatant ammonium sulfate was added to a finalconcentration of 1.5 M and the solution allowed to equilibrateovernight. After clarification the supernatant was loaded onto aphenyl-5PW column (GE Healthcare, Piscataway, N.J.) equilibrated in 1.5M ammonium sulfate, 20 mM Tris, pH 8.0. The column was washed with 4column volumes (CV), and IPD090Aa (TR1) polypeptide (SEQ ID NO: 10)containing fractions eluted with a 10 column volume gradient to 20 mMTris, pH 8.0. Eluate fractions containing IPD090Aa (TR1) polypeptide(SEQ ID NO: 10) were concentrated and desalted into PBS buffer using aSephadex™ G-25 (GE Healthcare) column and was submitted to bioassayagainst WCRW. The average WCRW scores for the IPD090Aa (TR1) polypeptide(SEQ ID NO: 10) dilution series from 8 assay replicates are shown inTable 4.

TABLE 4 Polypeptide Purified Concentration Polypeptide (mg/ml) Avg. WCRWScore IPD090Aa(TR1) 2.667 2.8 SEQ ID NO: 10 1.43 2.3 0.655 2.0 0.298 1.50.14 0.4 0.069 0 0.035 0 0.024 0 PBS buffer 0 0 control

Example 7—E. coli Expression of IPD090Ca Polypeptide

The sequence encoding the IPD090Ca polypeptide (SEQ ID NO: 6) wasisolated from strain JH23959-1 using primers CTB60-FOR (SEQ ID NO: 358)and CTB63-REV (SEQ ID NO: 359) to amplify the gene from strain JH23959-1and clone the IPD090Ca coding sequence (SEQ ID NO: 5) (with the nativestop codon (TAA)) into pET-24a (Novagen) for translation and pET-14b(Novagen) for an N-terminal translation of a 6×-His tag using NdeI/BamHIsites. The KOD Hot Start Master Mix (EMD Biosciences, San Diego, Calif.)was used for PCR amplification of the IPD090Ca gene on a Bio-Rad™ C1000Touch™ thermal cycler. Cycling parameters are as follows: 1 cycle at 95°C. for 2 minutes; 35 cycles of 95° C. for 20 seconds, 60° C. for 10seconds and 70° C. for 15 seconds; 1 cycle at 70° C. for 2 minutes.Amplicons were gel purified, ligated (T4 DNA Ligase, New EnglandBioLabs, Ipswich, Mass.) into expression vectors (as described above),transformed into E. coli One Shot® TOP10 high efficiency chemicallycompetent cells (Invitrogen) and clones were confirmed by sequencing.

Confirmed clones expressing IPD090Ca (SEQ ID NO: 5) in pET-24a/BL21, 50mL LB-CARB and KAN cultures were seeded with 500 μL of overnight cultureand incubated 37° C., 200 rpm until a OD₆₀₀ ˜0.8 was reached. Cultureswere induced with 0.3 mM IPTG and incubated at 16° C., 100 rpm overnight(˜20 hrs.). After induction, the cultures were centrifuged at 5,000 rcffor 15 minutes to pellet cells. The cell pellets were stored at −80° C.overnight prior to lysis. After freeze/thaw, the cell pellets were lysedwith 3 mL of lysis buffer (20 mM Tris pH 8, ¼×B-PER™ II, Omni-Cleave™,Ready-Lyse™ and Halt™ Protease Inhibitors), rocking at room temperaturefor 1 hour. The cell lysates were cleared via centrifugation at 13,000rcf for 10 minutes. 2.5 mL of each cleared lysate was applied to a PD10desalting column (GE Healthcare, Piscataway, N.J.), equilibrated withPBS. IPD090Ca polypeptide (SEQ ID NO: 6) was eluted off the PD10 columnswith 3.5 mL PBS and the lysate was submitted to bioassay against WCRW.The average WCRW score for the dilution series of IPD090Ca polypeptide(SEQ ID NO: 6) from 8 assay replicates are shown in Table 5.

TABLE 5 Total Lysate Protein Conc. Test Sample (mg/mL) Avg. WCRW ScoreIPD090Ca 3.000 2.5 (SEQ ID NO: 6) 2.060 2.5 1.242 2.9 0.691 2.5 0.3322.1 0.212 2.0 0.042 1.4 0.038 0 PBS Buffer 0 0 Control

Example 8—E. coli Expression of IPD090Fa Polypeptide

The IPD090Fa amino acid sequence (SEQ ID NO: 8) was identified by BLAST™search of the public non-redundant protein sequence database at NCBI(NCBI Reference Sequence: WP_019961352.1). The corresponding E. colioptimized coding sequence (SEQ ID NO: 7) was generated as twooverlapping synthetic DNA fragments (IDT, Coralville Iowa), the ends ofwhich contained 30 nucleotides of homology with pET-24a (Novagen) at theNdeI/XhoI sites. The IPD090Fa C-terminal 6×-His (SEQ ID NO: 350)expression vector was generated using NEBuilder™ (New England Biolabs,Ipswich Mass.). Positive clones were confirmed by DNA sequencing.

The IPD090Fa C-terminal 6×-His (SEQ ID NO: 350) expression construct wastransformed into E. coli BL21 (DE3, Agilent, Santa Clara Calif.)expression cells. 250 ml Luria Broth cultures (containing kanamycin)were grown at 37° C. until an OD600 of approximately 0.6 was reached andthen the cultures were induced with 1 mMisopropyl-β-D-1-thiogalactopyranoside (IPTG) and allowed to grow for anadditional 18 hours at 16° C., 250 rpm. The cultures were centrifuged at5,000 rcf for 15 minutes to pellet the cells. Cell pellets were lysedwith ¼ B-PER™ II reagent (Thermo Scientific), 20 mM Tris pH 8.0,OmniCleave™ endonuclease (Epicentre), ReadyLyse™ lysozyme (Epicentre)and HALT™ Protease Inhibitor Cocktail V (Millipore) for 120 minutesrocking at 30° C. The lysates were cleared via centrifugation at 13,000rcf for 10 minutes and the supernatants were brought up to 10 mMImidazole and then applied to separate 1 mL Ni-NTA (QIAGEN® Inc.,Valencia, Calif. 91355) columns equilibrated with Tris buffered saline(TBS), 10 mM imidazole. Columns were washed two times with 5 mL of 10 mMImidazole in TBS. Recombinantly expressed IPD090Fa C-terminal 6×-Hispolypeptide (SEQ ID NO: 351) was eluted off the columns with 1.2 mL of300 mM imidazole in TBS. The 1.2 mL eluate was applied to a Zeba SpinDesalting Column (Thermo) and buffer exchanged to TBS. Purified anddesalted IPD090Fa C-Terminal 6×-His tagged polypeptide (SEQ ID NO: 351)was submitted to bioassay against WCRW and was active (Table 6).

A cell pellet from a 1 L E. coli culture expressing the IPD090Fapolypeptide (SEQ ID NO: 8) was suspended in 5× volume (volume to weight)20 mM Tris pH 8.0+1:100 HALT™ proteinase inhibitor cocktail (Thermo).Cells were lysed at 25,000 PSI and the lysate centrifuged at 30,000 gfor 30 min. To the supernatant an equal volume of 3 M ammonium sulfatewas added dropwise while stirring to a final concentration of 1.5 M andthe solution was allowed to stir for at least 30 minutes. Afterclarification the supernatant was loaded onto a Phenyl-5PW column (TosohBioscience, King of Prussia, Pa.) equilibrated in 1.5 M ammoniumsulfate, 20 mM Tris, pH 8.0. The column was washed with 4 column volumes(CV), and IPD090Fa polypeptide (SEQ ID NO: 8) containing fractionseluted with a 15 CV gradient to 20 mM Tris, pH 8.0. Eluate with IPD090Fapolypeptide (SEQ ID NO: 8) was loaded onto a Mono Q™ column (GEHealthcare, Piscataway, N.J.) equilibrated in 20 mM Tris pH 8.0 bufferand IPD090Fa containing fractions eluted with a 40 CV gradient to 0.5 MNaCl, 20 mM Tris pH 8.0, concentrated, and further purified by sizeexclusion chromatography on an Superdex™ 200 column (GE Healthcare,Piscataway, N.J.) equilibrated in PBS. Based on SDS-PAGE, fractions withpurified IPD090Fa polypeptide (SEQ ID NO: 8) were combined.

Example 9—Diet-Based Bioassays with Corn Rootworm for Determination ofLC50 and IC50

Standardized corn rootworm diet incorporation bioassays were utilized totest the activity of the IPD090Aa polypeptide (SEQ ID NO: 2) on WCRW.Corn rootworm diet was prepared according to manufacturer's guidelinefor Diabrotica diet (Frontier, Newark, Del.). The test involved sixdifferent IPD090Aa polypeptide (SEQ ID NO: 2) doses plus buffer controlwith 32 observations for each dose in each bioassay. Neonates wereinfested into 96-well plates containing a mixture of the IPD090Aapolypeptide (SEQ ID NO: 2) (5 μL/well) and diet (25 μL/well), each wellwith approximately 5 to 8 larvae (<24 h post hatch). After one day asingle larva was transferred into each well of a second 96-well platecontaining a mixture of the IPD090Aa polypeptide (SEQ ID NO: 2) (20μL/well) and diet (100 μL/well) at the same concentration as thetreatment to which the insect was exposed on the first day. The plateswere incubated at 27° C., 65% RH in the dark for 6 days. The 50% lethalconcentration for polypeptides in the bioassay was calculated using“Dose Response Add-In for Excel” based on Probit analysis. Mortality andsevere stunted counts were scored and pooled as total response for thecalculation of inhibition of 50% of the individuals using the samemethod. The LC50 and IC50 against WCRW (Diabrotica virgifera virgifera)were 16.3 ppm and 7.4 ppm, respectively and against NCRW (Diabroticabarberi) were 35.6 ppm and 13 ppm, respectively. Against Diabroticaspeciosa the LC50 was >400 ppm and IC50=320 ppm. The same assay protocolwas used to evaluate the toxicity of IPD090Aa C-terminal 6×-Hispolypeptide (SEQ ID NO: 349) and IPD090Fa C-terminal 6×-His polypeptide(SEQ ID NO: 351) against WCRW and NCRW. The results are shown in Table6.

TABLE 6 Lower Upper Insect Sample LC/IC ppm 95% CL 95% CL Slope N WCRWIPD090Aa LC50 42.0 31.8 63.9 2.1 128.0 C-term-6xHis IC50 17.6 14.2 21.92.6 158.0 (SEQ ID NO: 349) IPD090Fa LC50 9.0 7.0 11.3 2.4 186.0C-term-6xHis IC50 5.7 4.4 7.0 2.7 154.0 (SEQ ID NO: 351) NCRW IPD090AaLC50 100.2 69.7 122.6 7.9 47.0 C-term-6xHis IC50 54.4 36.4 81.7 2.6 79.0(SEQ ID NO: 349) IPD090Fa LC50 13.9 10.5 18.2 3.4 79.0 C-term-6xHis IC509.2 6.5 12.0 4.0 63.0 (SEQ ID NO: 351)

Example 10—Testing Cross-Resistance of mCry3A-Selected WCRW

LC50 was also determined for IPD090Aa polypeptide (SEQ ID NO: 2) againstWCRW resistant to mCry3A and compared to susceptible WCRW using the samemethod as diet-based bioassays on WCRW as described in Example 8 above.A WCRW strain resistant to mCry3A was developed by selections ontransgenic maize plants with high level of mCry3A expression (>10,000ng/mg of total soluble protein in T0 roots) and high efficacy on WCRW.The resistance ratio (RR) was >92-fold to mCry3A for the colony based onLC50 in a diet-based assay (Patent Publication No. US 20140033361). TheRR was calculated as follows: RR=(LC50 of resistant WCRW)/(LC50 ofsusceptible WCRW). Table 7 shows that the WCRW strain resistant tomCry3A was not cross-resistant (RR=1.4-fold) to IPD090Aa polypeptide(SEQ ID NO: 2).

TABLE 7 IPD090Aa Resistance WCRW (SEQ ID NO: 2), Slope Ratio colony n(μg/mL) 95% CL (SE) (RR) Control 230 25.4 19.3-34.5 1.8 (0.3) 1.0mCry3A- 240 35.3 26.5-46.5 2.3 (0.4) 1.4 res*

Example 11—Chimeras Between IPD090 Homologs

To generate active variants of IPD090Aa polypeptide (SEQ ID NO: 2) withdiversified sequences, chimera genes between IPD090Aa (SEQ ID NO: 1) andIPD090Ca (SEQ ID NO: 5) were generated by multi-PCR fragment overlapassembly. For this purpose the nucleotide sequence of IPD090Ca was codonharmonized to that of IPD090Aa making the DNA homology higher to allowfor the family shuffling and chimera construction. The codon modifiedIPD090Ca coding sequence has the nucleic acid sequence of SEQ ID NO:345. A total of seven IPD090Aa/IPD090Ca chimera polynucleotides wereconstructed and cloned into pET24a. Constructs were transformed intoBL21 DE3 and cultured in 48-well plates for protein expression. Celllysates were generated by B-PER® Protein Extraction Reagent from ThermoScientific (3747 N. Meridian Rd., Rockford, Ill. USA 61101) and screenedfor WCRW insecticidal activity. Table 8 shows the chimera proteinboundaries and the % sequence identity to IPD090Aa polypeptide (SEQ IDNO: 2).

TABLE 8 IPD090Aa IPD090Ca % Seq. (SEQ ID NO: 2) (SEQ ID NO: 6) identityto WCRW Chimera Designation polynucleotide N-term fragment C-termfragment IPD090Aa active Chimera 1 SEQ ID NO: 12 SEQ ID NO: 11 M1-A239R241-K483 88.6 Yes Chimera 2 SEQ ID NO: 14 SEQ ID NO: 13 M1-V296P297-K483 90.9 Yes Chimera 3 SEQ ID NO: 16 SEQ ID NO: 15 M1-G348D349-K483 93.8 Yes Chimera 4 SEQ ID NO: 18 SEQ ID NO: 17 M1-Q382A383-K483 94.8 Yes Chimera 5 SEQ ID NO: 20 SEQ ID NO: 19 M1-G422A423-K483 97.7 Yes Chimera 6 SEQ ID NO: 22 SEQ ID NO: 21 M1-K442I443-K483 98.8 Yes Chimera 7 SEQ ID NO: 24 SEQ ID NO: 23 M1-Q144S146-K483 86.5 Yes

Example 12—IPD090Aa Variants with Multiple Amino Acid Substitutions

To create variants of IPD090Aa polypeptide (SEQ ID NO: 2) with multipleamino acid changes, variant libraries were generated by family shuffling(Chia-Chun J. Chang et al, 1999, Nature Biotechnology 17, 793-797)polynucleotides encoding IPD090Aa (SEQ ID NO: 2), and IPD090Ca (SEQ IDNO: 6).

Three libraries were constructed for generating IPD090Aa variants. Inthe first library, the polynucleotide sequences of SEQ ID NO: 1 and SEQID NO: 5 were used as library parents. In the second library, thepolynucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 5 were amplifiedin seven fragments with overlapping homology. Primers used to amplifythe fragments are summarized in Table 9. The overlapping fragments werepooled and assembled.

TABLE 9 Primer Sequence 90Aa Frag1GAA GGA GAT ATA CAT ATG GAA MAC RTA GAC TTG CCA CAR GGA CTT ForwardGTA AAC TTT TCC (SEQ ID NO: 360) 90Ca Frag1GAA GGA GAT ATA CAT ATG GAA MAC RTC GAC CTG CCG ACR GGA CTC ForwardGTC AAA TTT TCC (SEQ ID NO: 361) 90-2 ForwardTC GTR CCS GAG ATC GTC GAC GTS CAR CAG AAY GAC AGC GCM ASCTAC ACC AAC (SEQ ID NO: 362) 90-2 RCGTT GGT GTA GST KGC GCT GTC RTT CTG YTG SAC GTC GAC GAT CTC(SEQ ID NO: 363) 90-3 ForwardAAC GAG TTC CAC YCG YAT YCA GCA ATC GAT CAA CCT CTG GTC G(SEQ ID NO: 364) 90-3 RCACC GAA GGC ARG CGC ARC GAC CAG AGG TTG ATC GAT TGC TG (SEQ ID NO: 365)90-4 Forward ACC GGC ATC GTR ATG GGY GGM CGR GCC ATM CTC GCC KCC TCG GACCAA C (SEQ ID NO: 366) 90-4 RCGTT GGT CCG AGG MGG CGA GKA TGG CYC GKC CRC CCA TYA CGA TGCCGG T (SEQ ID NO: 367) 90-5 ForwardTTC CAG GCC TGG GTM GAC AGY GTG RGC RCC TCG CCS GAY TTC GTCGAY TTC GTY CCC ACC ATC CC (SEQ ID NO: 368) 90-5 RCGGG ATG GTG GGR ACG AAR TCG ACG AAR TCS GGC GAG GYG CYC ACRCTG TCK ACC CAG GCC TGG AA (SEQ ID NO: 369) 90-6 ForwardTAC GAC CTC AAT GCC GG (SEQ ID NO: 370) 90-6 RCCCG GCA TTG AGG TCG TA (SEQ ID NO: 371) 90-7 ForwardTAC AAC ACC GAY ACC GCR ATC AAC AAG (SEQ ID NO: 372) 90-7 RCCTT GTT GAT YGC GGT RTC GGT GTT GTA (SEQ ID NO: 372) 90Aa Frag 8CTC AGT GGT GGT GGT GGT GGT GCT CGA GCT ACT TGC CTA CGA AGG ReverseTAC AGG CAT AGA TG (SEQ ID NO: 374) 90Ca Frag 8CTC AGT GGT GGT GGT GGT GGT GCT CGA GTT ACT TGC CGA CGA AAG ReverseTGC AGG CAT AGA TG (SEQ ID NO: 375)

In the third library the native polynucleotide sequence (SEQ ID NO: 1)encoding the IPD090Aa polypeptide (SEQ ID NO: 2) and an E. coli codonoptimized polynucleotide sequence (SEQ ID NO: 345) encoding the IPD090Capolypeptide (SEQ ID NO: 6), were used as library parents.

After transforming the library variants into E. coli cells, the colonieswere picked and cultured in 48-well plates for protein expression. Celllysates were generated by B-PER® Protein Extraction Reagent from ThermoScientific (3747 N Meridian Rd, Rockford, Ill. USA 61101) and screenedfor WCRW insecticidal activity. The active variants were sequenced andthe amino acids substitutions were identified. In Library 1, 144variants were screened and 11 active unique variants were sequenceidentified. In Library 2, 96 variants were screened and 10 active uniquevariants were sequence identified. In Library 3, 168 variants werescreened and 64 active unique variants were sequence identified.

Percent sequence identity of active IPD090Aa variants to the IPD090Aapolypeptide (SEQ ID NO: 2) was calculated using the Needleman-Wunschalgorithm, as implemented in the Needle program (EMBOSS tool suite). Thepercent identity compared to the IPD090Aa polypeptide (SEQ ID NO: 2),variant designation, nucleotide sequences, and amino acid sequences ofthe resulting active IPD090Aa polypeptide variants are shown in Table10. Table 11 summarizes the % identity of the active variants comparedto IPD090Aa polypeptide (SEQ ID NO: 2), the number of variants with eachpercent identity, and the variant identification.

TABLE 10 % Identity to IPD090Aa (SEQ ID NO: 2) Variant PolynucleotidePolypeptide 90.5 S04515584 SEQ ID NO: 25 SEQ ID NO: 114 84.1 S04515608SEQ ID NO: 26 SEQ ID NO: 115 89.9 S04515618 SEQ ID NO: 27 SEQ ID NO: 11680.2 S04515626 SEQ ID NO: 28 SEQ ID NO: 117 81.6 S04515631 SEQ ID NO: 29SEQ ID NO: 118 82.2 S04515638 SEQ ID NO: 30 SEQ ID NO: 119 79.8S04515642 SEQ ID NO: 31 SEQ ID NO: 120 81.8 S04515648 SEQ ID NO: 32 SEQID NO: 121 94.2 S04515711 SEQ ID NO: 33 SEQ ID NO: 122 80.8 S04515723SEQ ID NO: 34 SEQ ID NO: 123 80 S04515724 SEQ ID NO: 35 SEQ ID NO: 12492.1 S04519420 SEQ ID NO: 36 SEQ ID NO: 125 84.1 S04519434 SEQ ID NO: 37SEQ ID NO: 126 83.9 S04519435 SEQ ID NO: 38 SEQ ID NO: 127 93.4S04519439 SEQ ID NO: 39 SEQ ID NO: 128 87.8 S04519446 SEQ ID NO: 40 SEQID NO: 129 83.9 S04519447 SEQ ID NO: 41 SEQ ID NO: 130 89 S04519473 SEQID NO: 42 SEQ ID NO: 131 96.7 S04519475 SEQ ID NO: 43 SEQ ID NO: 13294.2 S04519477 SEQ ID NO: 44 SEQ ID NO: 133 83.3 S04519504 SEQ ID NO: 45SEQ ID NO: 134 96.9 S04529311 SEQ ID NO: 46 SEQ ID NO: 135 90.7S04529312 SEQ ID NO: 47 SEQ ID NO: 136 89.6 S04529313 SEQ ID NO: 48 SEQID NO: 137 88 S04529314 SEQ ID NO: 49 SEQ ID NO: 138 88.4 S04529317 SEQID NO: 50 SEQ ID NO: 139 89.9 S04529318 SEQ ID NO: 51 SEQ ID NO: 140 93S04529319 SEQ ID NO: 52 SEQ ID NO: 141 92.4 S04529320 SEQ ID NO: 53 SEQID NO: 142 96.1 S04529325 SEQ ID NO: 54 SEQ ID NO: 143 93.6 S04529326SEQ ID NO: 55 SEQ ID NO: 144 91.7 S04529329 SEQ ID NO: 56 SEQ ID NO: 14593.6 S04529331 SEQ ID NO: 57 SEQ ID NO: 146 95.4 S04529338 SEQ ID NO: 58SEQ ID NO: 147 96.3 S04529347 SEQ ID NO: 59 SEQ ID NO: 148 94.8S04529348 SEQ ID NO: 60 SEQ ID NO: 149 89.9 S04529351 SEQ ID NO: 61 SEQID NO: 150 89.2 S04529352 SEQ ID NO: 62 SEQ ID NO: 151 86 S04529353 SEQID NO: 63 SEQ ID NO: 152 97.3 S04529355 SEQ ID NO: 64 SEQ ID NO: 15386.2 S04529359 SEQ ID NO: 65 SEQ ID NO: 154 88.8 S04529361 SEQ ID NO: 66SEQ ID NO: 155 95.9 S04529363 SEQ ID NO: 67 SEQ ID NO: 156 86.8S04529365 SEQ ID NO: 68 SEQ ID NO: 157 88 S04529371 SEQ ID NO: 69 SEQ IDNO: 158 90.5 S04529372 SEQ ID NO: 70 SEQ ID NO: 159 97.3 S04529374 SEQID NO: 71 SEQ ID NO: 160 96.5 S04529375 SEQ ID NO: 72 SEQ ID NO: 16183.9 S04529376 SEQ ID NO: 73 SEQ ID NO: 162 95.2 S04529377 SEQ ID NO: 74SEQ ID NO: 163 96.5 S04529378 SEQ ID NO: 75 SEQ ID NO: 164 85.1S04529380 SEQ ID NO: 76 SEQ ID NO: 165 94.2 S04529383 SEQ ID NO: 77 SEQID NO: 166 92.1 S04529386 SEQ ID NO: 78 SEQ ID NO: 167 94 S04529390 SEQID NO: 79 SEQ ID NO: 168 86 S04529393 SEQ ID NO: 80 SEQ ID NO: 169 86.2S04529396 SEQ ID NO: 81 SEQ ID NO: 170 92.8 S04529397 SEQ ID NO: 82 SEQID NO: 171 94.2 S04529401 SEQ ID NO: 83 SEQ ID NO: 172 90.3 S04529402SEQ ID NO: 84 SEQ ID NO: 173 92.3 S04529404 SEQ ID NO: 85 SEQ ID NO: 17490.3 S04529407 SEQ ID NO: 86 SEQ ID NO: 175 95 S04529410 SEQ ID NO: 87SEQ ID NO: 176 97.5 S04529419 SEQ ID NO: 88 SEQ ID NO: 177 99.4S04529422 SEQ ID NO: 89 SEQ ID NO: 178 95.2 S04529423 SEQ ID NO: 90 SEQID NO: 179 95.2 S04529426 SEQ ID NO: 91 SEQ ID NO: 180 90.3 S04529432SEQ ID NO: 92 SEQ ID NO: 181 91.1 S04529434 SEQ ID NO: 93 SEQ ID NO: 18293.6 S04529436 SEQ ID NO: 94 SEQ ID NO: 183 91.3 S04529437 SEQ ID NO: 95SEQ ID NO: 184 93.6 S04529443 SEQ ID NO: 96 SEQ ID NO: 185 98.3S04529446 SEQ ID NO: 97 SEQ ID NO: 186 89.9 S04529447 SEQ ID NO: 98 SEQID NO: 187 96.5 S04529455 SEQ ID NO: 99 SEQ ID NO: 188 97.7 S04529458SEQ ID NO: 100 SEQ ID NO: 189 92.1 S04529460 SEQ ID NO: 101 SEQ ID NO:190 91.3 S04529461 SEQ ID NO: 102 SEQ ID NO: 191 93.8 S04529462 SEQ IDNO: 103 SEQ ID NO: 192 88 S04529463 SEQ ID NO: 104 SEQ ID NO: 193 93.6S04529469 SEQ ID NO: 105 SEQ ID NO: 194 87.4 S04529471 SEQ ID NO: 106SEQ ID NO: 195 87.4 S04529479 SEQ ID NO: 107 SEQ ID NO: 196 91.1S04529481 SEQ ID NO: 108 SEQ ID NO: 197 89.4 S04529483 SEQ ID NO: 109SEQ ID NO: 198 87 S04529486 SEQ ID NO: 110 SEQ ID NO: 199 92.1 S04529493SEQ ID NO: 111 SEQ ID NO: 200 95.2 S04529495 SEQ ID NO: 112 SEQ ID NO:201 87.4 S04529498 SEQ ID NO: 113 SEQ ID NO: 202

TABLE 11 % Iden. to IPD090Aa (SEQ ID NO: 2) # variants Variants 99 1S04529422 98 1 S04529446 97 4 S04529458, S04529419, S04529355, S0452937496 7 S04529311, S04519475, S04529375, S04529378, S04529455, S04529347,S04529325 95 7 S04529363, S04529338, S04529377, S04529423, S04529426,S04529495, S04529410 94 6 S04529348, S04515711, S04519477, S04529383,S04529401, S04529390 93 8 S04529462, S04529326, S04529331, S04529436,S04529443, S04529469, S04519439, S04529319 92 7 S04529397, S04529320,S04529404, S04519420, S04529386, S04529460, S04529493 91 5 S04529437,S04529481, S04529329, S04529434, S04529461 90 6 S04529312, S04515584,S04529372, S04529402, S04529407, S04529432 89 8 S04515618, S04529318,S04529351, S04529447, S04529313, S04529483, S04529352, S04519473 88 5S04529361, S04529317, S04529314, S04529371, S04529463 87 5 S04519446,S04529471, S04529479, S04529498, S04529486 86 5 S04529365, S04529359,S04529396, S04529353, S04529393 85 1 S04529380 84 2 S04515608, S0451943483 4 S04519435, S04519447, S04529376, S04519504 82 1 S04515638 81 2S04515648, S04515631 80 3 S04515723, S04515626, S04515724 79 1 S04515642

Example 13—IPD090Aa Variants with Modified Physical Properties

A series of variants of the IPD090Aa polypeptide (SEQ ID NO: 2) withmodified physical properties were created by mutagenesis methods usingthe QuikChange™ Multi Site-Directed Mutagenesis Kit (Agilent).Oligonucleotides were designed and pooled to introduce conservative I toL and Y to F amino acid changes at selected positions within theIPD090Aa polypeptide (SEQ ID NO: 2). The library was expressed in E.coli and 204 isolates were screened as cleared lysates for WCRWinsecticidal activity. 71 unique WCRW active clones were identified andare summarized in Table 12.

TABLE 12 Variant Polynucleotide Polypeptide Amino acid sub. compared toIPD090Aa (SEQ ID NO: 2) S04509867 SEQ ID NO: 203 SEQ ID NO: 274 I038LS04509903 SEQ ID NO: 204 SEQ ID NO: 275 I004L, I038L, I375L S04509914SEQ ID NO: 205 SEQ ID NO: 276 I340L S04509946 SEQ ID NO: 206 SEQ ID NO:277 I375L S04513757 SEQ ID NO: 207 SEQ ID NO: 278 I080L S04537215 SEQ IDNO: 208 SEQ ID NO: 279 I080L, Y321F, Y333F, Y434F, I446L, I453LS04537217 SEQ ID NO: 209 SEQ ID NO: 280 I080L, I099L, Y321F, Y333F,I353L, Y434F, I453L S04537221 SEQ ID NO: 210 SEQ ID NO: 281 I080L,Y091F, I099L, I353L, I440L, Y457F S04537226 SEQ ID NO: 211 SEQ ID NO:282 I080L, Y333F, I340L, I446L, Y457F S04537227 SEQ ID NO: 212 SEQ IDNO: 283 I080L, I099L, Y333F, Y434F S04537230 SEQ ID NO: 213 SEQ ID NO:284 I080L, Y091F, Y339F, I353L, I440L S04537235 SEQ ID NO: 214 SEQ IDNO: 285 I080L, Y091F, Y321F, Y333F, I340L, I346L, I362L, Y434F, I440LS04537237 SEQ ID NO: 215 SEQ ID NO: 286 I080L, Y333F, Y434F S04537243SEQ ID NO: 216 SEQ ID NO: 287 I080L, I099L, Y339F, Y457F S04537244 SEQID NO: 217 SEQ ID NO: 288 I080L, Y091F, Y333F, I362L, I446L S04537246SEQ ID NO: 218 SEQ ID NO: 289 I080L, Y333F, I362L S04537249 SEQ ID NO:219 SEQ ID NO: 290 I080L, Y091F, Y333F, I440L S04537256 SEQ ID NO: 220SEQ ID NO: 291 I080L, Y091F, 1099L, Y321F, Y333F S04537260 SEQ ID NO:221 SEQ ID NO: 292 I080L, I099L, E331K, K332V, Y333P, R334G, V335Q,K336G, A337, N338, Y339, I340, D341, Q342, L343, V344, V345, I346, T347,G348, G349, S350, S351, T352, I353, E354, P355, P356, V357, G358, Y359,S360, K361, I362, E363, Y364, D365, L366, N367, A368, G369, A370, G371,G372, D373, F374, I375, Y376, L377, C378, Y379, H380, E381, Q382, T383,W384, Q385, A386, D387, R388, P389, K390, D391, A392, V393, T394, D395,I396, R397, I398, I399, F400, N401, K402, E403, P404, T405, P406, P407,G408, Y409, T410, K411, L412, P413, Q414, D415, L416, N417, K418, G419,A420, G421, G422, D423, D424, V425, F426, L427, C428, Y429, K430, T431,E432, A433, Y434, N435, T436, D437, T438, A439, I440, N441, K442, V443,T444, V445, I446, G447, G448, N449, N450, A451, D452, I453, N454, A455,P456, Y457, G458, Y459, L460, K461, V462, P463, G464, D465, L466, N467,R468, G469, A470, G471, G472, N473, F474, I475, Y476, A477, C478, T479,F480, V481, G482 S04537262 SEQ ID NO: 222 SEQ ID NO: 293 I362L, I440LS04537263 SEQ ID NO: 223 SEQ ID NO: 294 I080L, Y091F, Y333F, I362LS04537266 SEQ ID NO: 224 SEQ ID NO: 295 Y091F, Y321F, I440L S04537271SEQ ID NO: 225 SEQ ID NO: 296 I080L, Y333F, I346L, Y434F, I440LS04537273 SEQ ID NO: 226 SEQ ID NO: 297 I080L, I099L, Y321F, Y333F,I362L, I440L S04537275 SEQ ID NO: 227 SEQ ID NO: 298 I080L, Y339FS04537281 SEQ ID NO: 228 SEQ ID NO: 299 I080L, I099L, Y333F, I353L,Y434F, I446L S04537282 SEQ ID NO: 229 SEQ ID NO: 300 I080L, Y333F, I440LS04537285 SEQ ID NO: 230 SEQ ID NO: 301 I080L, Y091F, Y333F, I346L,I440L, Y457F S04537286 SEQ ID NO: 231 SEQ ID NO: 302 I080L, I340L,I362L, Y434F S04537292 SEQ ID NO: 232 SEQ ID NO: 303 I080L, I340L,I346L, I362L, Y434F S04537293 SEQ ID NO: 233 SEQ ID NO: 304 I080L,I099L, Y333F, I353L S04537294 SEQ ID NO: 234 SEQ ID NO: 305 I080L,I099L, I346L, I362L, I440L S04537296 SEQ ID NO: 235 SEQ ID NO: 306I080L, Y091F, I099L, Y321F, Y333F, I340L, S350I, S351Q, T352P, I353S,E354N, P355H, P356R, V357S, G358A, Y359T, S360A, K361R, I362S, E363S,Y364T, D365T, L366S, N367M, A368P, G369V, A370P, G371A, G372V, D373T,F374S, I375S, Y376T, L377C, C378A, Y379I, H380T, E381N, Q382K, T383P,W384G, Q385R, A386P, D387T, R388G, P389L, D391M, A392L, V393, T394,D395, I396, R397, I398, I399, F400, N401, K402, E403, P404, T405, P406,P407, G408, Y409, T410, K411, L412, P413, Q414, D415, L416, N417, K418,G419, A420, G421, G422, D423, D424, V425, F426, L427, C428, Y429, K430,T431, E432, A433, Y434, N435, T436, D437, T438, A439, I440, N441, K442,V443, T444, V445, I446, G447, G448, N449, N450, A451, D452, I453, N454,A455, P456, Y457, G458, Y459, L460, K461, V462, P463, G464, D465, L466,N467, R468, G469, A470, G471, G472, N473, F474, I475, Y476, A477, C478,T479, F480, V481, G482 S04537298 SEQ ID NO: 236 SEQ ID NO: 307 I080L,I340L, I362L, D437A, T438P, A439H, I440S, N441T, K442R, V443S, T444R,V445S, I446S, G447A, G448A, N449T, N450M, A451R, D452I, I453S, N454T,A455L, Y457L, G458V, Y459I, L460, K461, V462, P463, G464, D465, L466,N467, R468, G469, A470, G471, G472, N473, F474, I475, Y476, A477, C478,T479, F480, V481, G482 S04537300 SEQ ID NO: 237 SEQ ID NO: 308 Y333F,I340L, I362L S04537301 SEQ ID NO: 238 SEQ ID NO: 309 I080L, Y091F, I340LS04537303 SEQ ID NO: 239 SEQ ID NO: 310 Y091F, Y333F, I446L S04537304SEQ ID NO: 240 SEQ ID NO: 311 I080L, Y091F, Y333F, Y434F S04537305 SEQID NO: 241 SEQ ID NO: 312 Y091F, Y333F S04537309 SEQ ID NO: 242 SEQ IDNO: 313 I080L, Y091F, I099L, Y339F, I346L, I353L, Y434F, I440L, Y457FS04537312 SEQ ID NO: 243 SEQ ID NO: 314 I080L, Y091F, K319S, H320I,Y321S, D322M, D323T, V324S, W325G, A326R, P327R, A328R, Q329N, S330R,E331K, K332S, Y333S, R334G, V335S, K336R, A337L, N338T, Y339T, I340S,D341T, Q342N, L343W, V344W, V345S, I346S, T347P, G348A, G349V, S350V,S351Q, T352P, I353S, E354N, P355H, P356R, V357S, G358A, Y359T, S360A,K361S, I362S, E363S, Y364T, D365T, L366S, N367M, A368P, G369V, A370P,G371A, G372V, D373T, F374S, I375S, Y376T, L377C, C378A, Y379I, H380T,E381N, Q382K, T383P, W384G, Q385R, A386P, D387T, R388G, P389L, D391M,A392L, V393, T394, D395, I396, R397, I398, I399, F400, N401, K402, E403,P404, T405, P406, P407, G408, Y409, T410, K411, L412, P413, Q414, D415,L416, N417, K418, G419, A420, G421, G422, D423, D424, V425, F426, L427,C428, Y429, K430, T431, E432, A433, Y434, N435, T436, D437, T438, A439,I440, N441, K442, V443, T444, V445, I446, G447, G448, N449, N450, A451,D452, I453, N454, A455, P456, Y457, G458, Y459, L460, K461, V462, P463,G464, D465, L466, N467, R468, G469, A470, G471, G472, N473, F474, I475,Y476, A477, C478, T479, F480, V481, G482 S04537314 SEQ ID NO: 244 SEQ IDNO: 315 I080L, I099L, I346L, I440L S04537315 SEQ ID NO: 245 SEQ ID NO:316 I080L, Y091F, Y321F, Y333F S04537319 SEQ ID NO: 246 SEQ ID NO: 317I080L, I099L, Y333F, I362L, I453L S04537321 SEQ ID NO: 247 SEQ ID NO:318 Y091F, Y333F, I440L, Y457F S04537322 SEQ ID NO: 248 SEQ ID NO: 319I080L, Y091F, I099L, I346L, I362L, Y434F, I440L S04537325 SEQ ID NO: 249SEQ ID NO: 320 I080L, Y091F, 1099L, Y321F, I346L, Y434F, Y457F S04537326SEQ ID NO: 250 SEQ ID NO: 321 I080L, Y091F, I099L, A316P, M317C, R318A,K319S, H320I, Y321S, D322M, D323T, V324S, W325G, A326R, P327R, A328R,Q329N, S330R, E331K, K332S, Y333S, R334G, V335S, K336R, A337L, N338T,Y339T, I340S, D341T, Q342N, L343W, V344W, V345S, I346S, T347P, G348A,G349V, S350V, S351Q, T352P, I353S, E354N, P355H, P356R, V357S, G358A,Y359T, S360A, K361R, I362S, E363S, Y364T, D365T, L366S, N367M, A368P,G369V, A370P, G371A, G372V, D373T, F374S, I375S, Y376T, L377C, C378A,Y379I, H380T, E381N, Q382K, T383P, W384G, Q385R, A386P, D387T, R388G,P389L, D391M, A392L, V393, T394, D395, I396, R397, I398, I399, F400,N401, K402, E403, P404, T405, P406, P407, G408, Y409, T410, K411, L412,P413, Q414, D415, L416, N417, K418, G419, A420, G421, G422, D423, D424,V425, F426, L427, C428, Y429, K430, T431, E432, A433, Y434, N435, T436,D437, T438, A439, I440, N441, K442, V443, T444, V445, I446, G447, G448,N449, N450, A451, D452, I453, N454, A455, P456, Y457, G458, Y459, L460,K461, V462, P463, G464, D465, L466, N467, R468, G469, A470, G471, G472,N473, F474, I475, Y476, A477, C478, T479, F480, V481, G482 S04537328 SEQID NO: 251 SEQ ID NO: 322 I080L, Y091F, Y321F, I340L, Y434F, I440LS04537330 SEQ ID NO: 252 SEQ ID NO: 323 I080L, Y091F, Y333F, I446LS04537332 SEQ ID NO: 253 SEQ ID NO: 324 I080L, Y091F, Y333F, Y457FS04537334 SEQ ID NO: 254 SEQ ID NO: 325 I080L, I340L, I440L, Y457FS04537337 SEQ ID NO: 255 SEQ ID NO: 326 I080L, Y091F, Y333F S04537339SEQ ID NO: 256 SEQ ID NO: 327 Y091F, Y333F S04537347 SEQ ID NO: 257 SEQID NO: 328 I080L, Y321F, Y333F, I440L, Y457F S04537349 SEQ ID NO: 258SEQ ID NO: 329 I080L, Y333F, I446L S04537350 SEQ ID NO: 259 SEQ ID NO:330 I080L, Y091F, Y333F, I446L S04537351 SEQ ID NO: 260 SEQ ID NO: 331I080L, I340L, I440L S04537352 SEQ ID NO: 261 SEQ ID NO: 332 Y091F,Y333F, I346L, Y434F, Y457F S04537359 SEQ ID NO: 262 SEQ ID NO: 333I080L, Y091F, Y339F, I346L, Y434F S04537360 SEQ ID NO: 263 SEQ ID NO:334 I080L, I099L, Y333F, I362L, Y434F S04537367 SEQ ID NO: 264 SEQ IDNO: 335 I080L, Y091F, Y333F S04537369 SEQ ID NO: 265 SEQ ID NO: 336I080L, Y091F, I099L, Y333F, Y339F, I346L, I353L, I362L, I440L S04537371SEQ ID NO: 266 SEQ ID NO: 337 I080L, Y091F, 1099L, Y321F, Y333F, Y339F,I346L, Y434F, I453L S04537373 SEQ ID NO: 267 SEQ ID NO: 338 I080L,Y321F, Y333F, Y434F, I446L S04537377 SEQ ID NO: 268 SEQ ID NO: 339I080L, Y091F, A239T, Y339F, I453L S04537385 SEQ ID NO: 269 SEQ ID NO:340 I080L, Y333F, Y434F, Y457F S04537389 SEQ ID NO: 270 SEQ ID NO: 341I080L, Y321F, Y333F, I453L S04537400 SEQ ID NO: 271 SEQ ID NO: 342I080L, Y091F, Y333F, I353L S04537401 SEQ ID NO: 272 SEQ ID NO: 343I080L, I099L, Y333F, I353L, I440L S04537402 SEQ ID NO: 273 SEQ ID NO:344 I080L, Y091F, Y333F, I446L

Example 14—Mode of Action

Bioactivity of purified recombinant protein incorporated into artificialdiet revealed toxicity of IPD090Aa polypeptide (SEQ ID NO: 2) to WCRWlarvae. To understand the mechanism of IPD090Aa polypeptide (SEQ ID NO:2) toxicity, specific binding of the purified protein with WCRW midguttissue was evaluated by in vitro competition assays. Midguts wereisolated from third instar WCRW larvae to prepare brush border membranevesicles (BBMV) following a method modified from Wolfersberger et al.(Comp Bioch Physiol 86A: 301-308, 1987) using amino-peptidase activityto track enrichment. BBMVs represent the apical membrane component ofthe epithelial cell lining of insect midgut tissue and therefore serveas a model system for how insecticidal proteins interact within the gutfollowing ingestion.

IPD090Aa polypeptide (SEQ ID NO: 2) was re-purified via anion exchangechromatography using a AKTA™ Purifier 10 (GE Life Sciences) with aFrac-950 fraction collector. An aliquot of purified IPD090Aa polypeptide(SEQ ID NO: 2) from Example 4 was taken from −80° C. storage anddialyzed 1 hr. at 4° C. against 20 mM CAPS pH 9.6 (‘Eluent A’) andloaded onto a 1 mL HiTrap™ Q FF column (GE Life Sciences) equilibratedin Eluent A. A 30 column volume gradient from 0 to 50% Eluent B (20 mMCAPS pH 9.6+1 M NaCl) at 1 mL/min was applied. Fractions near the apexof the elution peak were combined and dialyzed into Binding buffer (50mM sodium chloride, 2.7 mM potassium chloride, 8.1 mM disodium hydrogenphosphate, and 1.47 mM potassium dihydrogen phosphate, pH 7.5).

The purified IPD090Aa polypeptide (SEQ ID NO: 2) was labeled withAlexa-Fluor® 488 (Life Technologies) and unincorporated fluorophore wasseparated from labeled protein using buffer exchange resin (LifeTechnologies, A30006) following manufacturer's recommendations. Prior tobinding experiments, proteins were quantified by gel densitometryfollowing Simply Blue® (Thermo Scientific) staining of SDS-PAGE resolvedsamples that included BSA as a standard.

To demonstrate specific binding and to evaluate affinity, BBMVs (5 μg)were incubated with 6.3 nM of Alexa-labeled IPD090Aa polypeptide (SEQ IDNO: 2) in 100 μL of binding buffer for 1 hr. at RT in the absence andpresence of 13 μM of unlabeled IPD090Aa polypeptide (SEQ ID NO: 2).Centrifugation at 20,000×g was used to pellet the BBMVs to separateunbound IPD090Aa polypeptide (SEQ ID NO: 2) remaining in solution. TheBBMV pellet was then washed twice with binding buffer to eliminateremaining unbound IPD090Aa polypeptide (SEQ ID NO: 2). The final BBMVpellet (with bound fluorescent protein) was solubilized in reducingLaemmli sample buffer, heated to 100° C. for 5 minutes, and subjected toSDS-PAGE using 4-12% Bis-Tris polyacrylamide gels (Life Technologies).The amount of Alexa-labeled IPD090Aa polypeptide (SEQ ID NO: 2) in thegel from each sample was measured by a digital fluorescence imagingsystem (ImageQuant™ LAS4000-GE Healthcare). Digitized images wereanalyzed by densitometry software (Phoretix™ 1 D, TotalLab, Ltd.) FIG. 2shows that IPD090Aa polypeptide (SEQ ID NO: 2) binds specifically to 5μg of WCRW BBMVs.

Example 15—Vector Constructs for Expression of IPD090Aa Polypeptides inPlants

Plant expression vectors were constructed to include a transgenecassette containing two different gene designs encoding the IPD090polypeptide of SEQ ID NO: 377 and one gene design encoding the IPD090polypeptide of SEQ ID NO: 10 under control of the maize ubiquitinpromoter (Christensen, et al., 1992, Christensen and Quail 1996) andlinked to the PINII terminator (Keil et al., 1986, Nucleic AcidsResearch 14: 5641-5650; An et al., 1989, The Plant Cell 1: 115-122). Theresulting constructs, PHP73234, PHP73237 for the IPD090 polypeptide ofSEQ ID NO: 377 and PHP77372 for IPD090 polypeptide of SEQ ID NO: 10,were used to generate transgenic maize events to test for efficacyagainst corn rootworm provided by expression of these polypeptides.

Example 16—Agrobacterium-Mediated Transformation of Maize andRegeneration of Transgenic Plants

For Agrobacterium-mediated transformation of maize with the expressionvectors PHP73234, PHP73237, and PHP77372, the method of Zhao was used(U.S. Pat. No. 5,981,840 and PCT Patent Publication Number WO1998/32326; the contents of which are hereby incorporated by reference).Briefly, immature embryos were isolated from maize and the embryoscontacted with a suspension of Agrobacterium under conditions wherebythe bacteria are capable of transferring the PHP73234, PHP73237 andPHP77372 vectors to at least one cell of at least one of the immatureembryos (step 1: the infection step). In this step the immature embryoswere immersed in an Agrobacterium suspension for the initiation ofinoculation. The embryos were co-cultured for a time with theAgrobacterium (step 2: the co-cultivation step). The immature embryoswere cultured on solid medium following the infection step. Followingthis co-cultivation period an optional “resting” step is contemplated.In this resting step, the embryos were incubated in the presence of atleast one antibiotic known to inhibit the growth of Agrobacteriumwithout the addition of a selective agent for plant transformation (step3: resting step). The immature embryos were cultured on solid mediumwith antibiotic, but without a selecting agent, for elimination ofAgrobacterium and for a resting phase for the infected cells. Next,inoculated embryos were cultured on medium containing a selective agentand growing transformed callus is recovered (step 4: the selectionstep). The immature embryos were cultured on solid medium with aselective agent resulting in the selective growth of transformed cells.The callus was then regenerated into plants (step 5: the regenerationstep), and calli grown on selective medium or cultured on solid mediumto regenerate the plants.

For detection of the IPD090Aa polypeptide (SEQ ID NO: 2) and IPD090Aa(TR1) polypeptide (SEQ ID NO: 10) in leaf tissue 4 lyophilized leafpunches/sample were pulverized and resuspended in 100 μL PBS containing0.1% Tween 20 (PBST), 1% beta-mercaoptoethanol containing 1 tablet/7 mLcomplete Mini proteinase inhibitor (Roche 1183615301). The suspensionwas sonicated for 2 min and then centrifuged at 4° C., 20,000 g for 15min. To a supernatant aliquot ⅓ volume of 3× NuPAGE® LDS Sample Buffer(Invitrogen™ (CA, USA), 1% beta-mercaoptoethanol containing 1 tablet/7mL complete Mini proteinase inhibitor was added. The mixture was heatedat 80° C. for 10 min and then centrifuged. A supernatant sample wasloaded on 4-12% Bis-Tris Midi gels with MES running buffer as permanufacturer's (Invitrogen™) instructions and transferred onto anitrocellulose membrane using an iBlot® apparatus (Invitrogen™). Thenitrocellulose membrane was incubated in PBST containing 5% skim milkpowder for 2 hours before overnight incubation in affinity-purifiedrabbit anti-IPD090Aa (SEQ ID NO: 2) polyclonal antibody in PBSTovernight. The membrane was rinsed three times with PBST and thenincubated in PBST for 15 min and then two times 5 min before incubatingfor 2 hours in PBST with goat anti-rabbit-HRP for 3 hours. The detectedproteins were visualized using ECL Western Blotting Reagents (GEHealthcare cat #RPN2106) and visualized using a luminescent imageanalyzer (ImageQuant LAS 4000, GE Healthcare). For detection of theIPD090Aa polypeptide (SEQ ID NO: 2) and IPD090Aa (TR1) polypeptide (SEQID NO: 10) in roots the roots were lyophilized and 2 mg powder persample was resuspended in LDS, 1% beta-mercaptoethanol containing 1tablet/7 mL Complete Mini proteinase inhibitor was added. The mixturewas heated at 80° C. for 10 min and then centrifuged at 4° C., 20,000 gfor 15 min. A supernatant sample was loaded on 4-12% Bis-Tris Midi gelswith MES running buffer as per manufacturer's (Invitrogen™) instructionsand transferred onto a nitrocellulose membrane using an iBlot® apparatus(Invitrogen™). The nitrocellulose membrane was incubated in PBSTcontaining 5% skim milk powder for 2 hours before overnight incubationin affinity-purified polyclonal rabbit anti-IPD090Aa antibody in PBSTovernight. The membrane was rinsed three times with PBST and thenincubated in PBST for 15 min and then two times 5 min before incubatingfor 2 hours in PBST with goat anti-rabbit-HRP for 3 hrs. The antibodybound insecticidal proteins were detected using ECL™ Western BlottingReagents (GE Healthcare cat #RPN2106) and visualized using a luminescentimage analyzer (ImageQuant™ LAS 4000, GE Healthcare). Transgenic maizeplants positive for expression of the insecticidal proteins are testedfor pesticidal activity using standard bioassays known in the art. Suchmethods include, for example, root excision bioassays and whole plantbioassays. See, e.g., US Patent Application Publication Number US2003/0120054 and International Publication Number WO 2003/018810.

Example 17—Greenhouse Efficacy of IPD090 Polypeptide Events

T0 greenhouse efficacy results for events generated from PHP73234,PHP73237 and PHP77372 constructs are shown in FIG. 3. Efficacy forevents derived from all 3 constructs was observed relative to negativecontrol events (Empty) as measured by root protection from western cornrootworm. Root protection was measured according to the number of nodesof roots injured (CRWNIS=corn rootworm node injury score) using themethod developed by Oleson, et al. (2005) [J. Econ Entomol. 98(1):1-8].The root injury score is measured from “0” to “3” with “0” indicating novisible root injury, “1” indicating 1 node of root damage, “2”indicating 2 nodes or root damage, and “3” indicating a maximum score of3 nodes of root damage. Intermediate scores (e.g. 1.5) indicateadditional fractions of nodes of damage (e.g. one and a half nodesinjured). FIG. 3 shows that a large proportion of events from PHP73234,PHP73237 and PHP77372 performed better than the negative control andhave rootworm injury scores of <1.0.

Example 18—Three-Dimensional Structure of IPD090Aa as Determined byX-Ray Crystallography

Crystals of IPD090Aa variant 1167 were grown by hanging drop vapordiffusion method at 25° C. Crystals were obtained by mixing 2 ul of a 10mg/ml protein solution and 2 ul of crystallization solution containing0.2M MgCl₂ hexahydrate, 0.1M HEPES pH=7.5 and 30% PEG 400. Crystals weremounted in 0.5 mM loop and cryoprotected with the addition of ˜20%glycerol in the crystallization solution. They were flash frozen inliquid N₂ and mounted on a Rigaku Micromax-007 HF x-ray source at IowaState University Macromolecular X-ray Crystallography facility. 2.1 Ådata were collected using an R-Axis IV++ image plate detector at adistance of 165.0 mM. 60° of data were collected at 0.50 image width.Diffraction data was indexed and integrated with iMOSFILM (CCP4 GNULicense) (Battye, T. G. G, et al. (2011) Acta Cryst. D67, 271-281)(Steller, I et al. (1997) J. Appl. Cryst. 30, 1036-1040) and scaled withSCALA (Kabsch, W. 1998) J. Appl. Cryst. 21, 916-924. The structure wassolved using the molecular replacement program PhaserMR (McCoy, A. J. etal (2007) J. Appl. Cryst. 40, 658-674). The structure of aMACPF/perforin-like protein from Photorhabdus luminescens (PDB ID 2QP2)(Rosado, C. J. et al. (2007) Science 317, 1548-1551) was used as thesearch model. A suitable solution for the rotation and translationfunctions was identified. The sequence for IPD090Aa variant 1167 wasthen built into the electron density using WinCoot© (Emsley P, et al.(2010) ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 66,486-501). The model was refined using Refmac5 (Murshudov, G. et al.(1996) in the Refinement of Protein structures, Proceedings of DaresburyStudy Weekend;

Murshudov, G. N. et al. (1997) Acta Cryst. D53, 240-255) to anR-factor=0.236 and R-free=0.267 with >96% of amino acids in allowedregions of the Ramachandran Plot. Table 13 shows the data collection andrefinement statistics.

TABLE 13 Data collection Statistics Space Group P4₁2₁2 Resolution 2.13Cell Dimensions a b c α β γ 127.61 127.61 116.12 90 90 90 Reflections244629 R_(merge) 10.40% Completeness (%) 99.5 I/Sigmal 10.8 Multiplicity4.6 Refinement Statistics Resolution (Å) 2.13 No. reflections 51601R_(work)/R_(free) 21.45/24.54 No. Atoms Protein 3731 Water 184 Ligand 1B-factors(Å²) 32.84 R.M.S. deviations Bond Lengths(Å) 0.019 Bond Angles(°) 1.943 Ramachadran Plot Favored 95.62% Allowed  3.55% Outliers  0.84%Procheck Overall G-factor −0.1

The overall structure of IPD090Aa resembles that of other membraneattack complex/perforin (MACPF) containing proteins. Its N-terminaldomain is comprised of the MACPF domain while the C-terminal domaincontains the β-prism domain (FIG. 4). Secondary structures are labeledaccording to Rosado et al (2007 Science 317, 1548-1551). Mg+ atom isshown as a sphere at the bottom of the β-prism domain. The two clustersof helices (CH1 and CH2) are structurally similar to the transmembranehelices (TMH1 and TMH2) of cholesterol-dependent cytolysin (CDC) familyof toxins. The overall shape of the N-terminal MACPF domain is somewhatboxed shaped (˜42 Å×44 Å×24 Å) with a central L-shaped 4 strandedantiparallel β-sheet and 2 clusters of α-helices. The N-terminal 17amino acids in the MACPF domain form a 5^(th) member of the centralL-shaped β-sheet, but is parallel to strand 4 (FIG. 5). The MACPF domainfrom P. luminescens has an α-helical N-terminus. The C-terminal β-prismdomain is located at the bottom of and underneath the central β-sheet.It is connected to the MACPF domain through a five amino acid linkerthat adopts an extended β-strand-like conformation. The β-prism domainis made up of three β-stranded antiparallel β-sheets with a β-fold axisrunning through the center of the domain (FIG. 6). A Mg⁺² ion is locatedon this β-fold axis and coordinated by backbone carbonyl atoms fromL365, L415, L465 and sidechain carbonyl atoms of N366, N416, and N466.While a role for the Mg⁺² in insecticidal activity has not beenobserved, the Mg⁺² ion fills an anion hole at this location in themolecule and aides in maintaining the arrangement of the 3 antiparallelβ-sheets around the β-fold axis.

The above description of various illustrated embodiments of thedisclosure is not intended to be exhaustive or to limit the scope to theprecise form disclosed. While specific embodiments of and examples aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. The teachings providedherein can be applied to other purposes, other than the examplesdescribed above. Numerous modifications and variations are possible inlight of the above teachings and, therefore, are within the scope of theappended claims.

These and other changes may be made in light of the above detaileddescription. In general, in the following claims, the terms used shouldnot be construed to limit the scope to the specific embodimentsdisclosed in the specification and the claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, manuals, books or otherdisclosures) in the Background, Detailed Description, and Examples isherein incorporated by reference in their entireties.

Efforts have been made to ensure accuracy with respect to the numbersused (e.g. amounts, temperature, concentrations, etc.) but someexperimental errors and deviations should be allowed for. Unlessotherwise indicated, parts are parts by weight, molecular weight isaverage molecular weight; temperature is in degrees centigrade; andpressure is at or near atmospheric.

That which is claimed is:
 1. A DNA construct comprising a recombinantpolynucleotide operably linked to a heterologous regulatory element,wherein the recombinant polynucleotide encodes an insecticidalpolypeptide having at least 80% sequence identity to the amino acidsequence of SEQ ID NO:
 2. 2. A transgenic plant comprising the DNAconstruct of claim
 1. 3. A method of inhibiting growth or killing aninsect pest or pest population, comprising contacting the insect pestwith an insecticidal polypeptide having at least 80% sequence identityto the amino acid sequence of SEQ ID NO: 2, wherein the insecticidalpolypeptide is joined to a heterologous signal sequence or a transitsequence.
 4. The method of claim 3, wherein the insect pest or pestpopulation is resistant to at least one Cry insecticidal protein.
 5. Atransformed prokaryotic cell comprising the DNA construct of claim 1.